7 NUCLEIC ACIDS (AHL)
Introduction structure of DNA is ideally suited to its function.
Information transferred from DNA to mRNA is
The discovery of the structure of DNA translated into an amino acid sequence.
revolutionized biology. Information stored in a
coded form in DNA is copied onto mRNA. The
7.1 DNA structure and replication
Understanding Applications
DNA structure suggested a mechanism or DNA Rosalind Franklins and Maurice Wilkins
replication. investigation o DNA structure by X-ray
d i ra cti o n .
Nucleosomes help to supercoil the DNA.
DNA replication is continuous on the leading Tandem repeats are used in DNA profling.
Use o nucleotides containing
strand and discontinuous on the lagging strand.
DNA replication is carried out by a complex dideoxyribonucleic acid to stop DNA replication
in preparation o samples or base sequencing.
system o enzymes.
DNA polymerases can only add nucleotides to Skills
the 3 end o a primer. Analysis o results o the Hershey and Chase
Some regions o DNA do not code or proteins experiment providing evidence that DNA is the
genetic material.
but have other important unctions.
Utilization o molecular visualization sotware
Nature of science to analyse the association between protein and
DNA within a nucleosome.
Making careul observations: Rosalind
Franklins X-ray diraction provided crucial
evidence that DNA is a double helix.
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7 NUCLEIC ACIDS (AHL)
The HersheyChase experiment
Analysis of the results of the HersheyChase experiment providing evidence that
DNA is the genetic material.
From the late 1 800s, scientists were convinced Alred Hershey and Martha Chase wanted
that chromosomes played a role in heredity to ascertain whether the genetic material o
and that the hereditary material had a chemical viruses was protein or DNA. In the 1 950s, it was
nature. Aware that chromosomes were composed known that viruses are inectious particles which
o both protein and nucleic acid, both molecules transorm cells into virus-producing actories by
were contenders to be the genetic material. becoming bound to host cells and injecting their
Until the 1 940s, the view that protein was the genetic material. The non-genetic portion o the
hereditary material was avoured, as it was a virus remains outside the cell. An inected cell
class o macromolecules that had great variety then manuactures large numbers o new viruses
due to twenty naturally occurring sub-units as and bursts, releasing them to the environment
opposed to our nucleotide sub- units. Further, ( see fgure 1 ) . Viruses are oten specifc to a
many specifc unctions had been identifed or certain cell type. The virus they chose to work
proteins. Variety and specifcity o unction were with was the T2 bacteriophage because o its very
two properties that were expected to be essential simple structure. It has a coat composed entirely
requirements or the hereditary material. o protein while DNA is ound inside the coat.
DNA
protein
Figure 1 Coloured transmission electron micrograph (TEM) o T2 Figure 2 Diagram illustrating the structure o
viruses (blue) bound to an Escherichia coli bacterium. Each virus the T2 virus
consists o a large DNA-containing head and a tail composed o a
central sheath with several fbres. The fbres attach to the host cell
surace, and the virus DNA is injected into the cell through the sheath.
It instructs the host to build copies o the virus (blue, in cell)
Data-based questions: The HersheyChase experiment
Alred Hershey and Martha Chase were two phosphorus. They inected bacteria separately
scientists who worked to resolve the debate over with the two types o viruses. They used a
the chemical nature o the genetic material. blender to separate the non-genetic component
In their experiment, they took advantage o o the virus rom the cell and then centriuged
the act that DNA contains phosphorus but the culture solution to concentrate the cells in
not sulphur while proteins contain sulphur a pellet. The cells were expected to have the
but not phosphorus. They cultured viruses radioactive genetic component o the virus in
that contained proteins with radioactive (35S) them. They measured the radioactivity in the
sulphur and they separately cultured viruses pellet and the supernatant. Figure 3 represents
that contained DNA with radioactive (32P) the process and results o the experiment.
344
7.1 D N A s tr u ctu re AN D re pli c Ati o N
radioactive protein (35S)
protein coat with 35S virus radioactivity (35S) in supernatant
bacteria bacterium
radioactive DNA (32P)
DNA with 32P virus
bacteria bacterium
radioactivity (32P) in pellet
Questions % of isotope in supernatant percentage of isotope in supernatant after 8 minutes agitation
100%
a) Explain what a supernatant is.
80%
b) Explain why the genetic material should be
ound in the pellet and not the supernatant. 60%
c) Determine the percentage o the 32P that 40%
remains in the supernatant.
20%
d) Determine the percentage o 35S that remains
in the supernatant. 0% 32P
35S
e) Discuss the evidence that DNA is the chemical
which transorms the bacteria into inected Figure 3
cells.
X-ay dfan an a vdn ma
Making careul observations: Rosalind Franklins X-ray difraction provided crucial
evidence that DNA was a helix.
Two names are usually remembered in Another key fgure in the discovery o DNA was
connection with the discovery o DNA, Crick and Rosalind Franklin. In 1 950, she became a research
Watson. Flashes o insight led to their success, but associate in the biophysics unit at Kings C ollege,
they could not have achieved it without skilled London. The unit was already investigating the
experimental work and careul observations by structure o DNA by X-ray diraction. Franklin
other scientists. One o these was Erwin Charga. had already become skilled in techniques o
His research into the percentage base composition crystallography and X-ray diraction while
o DNA is described in the data-based question researching other carbon compounds at an
in sub-topic 2.6 (page 1 07) . institute in Paris.
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7 NUCLEIC ACIDS (AHL)
At Kings College she improved the resolution diraction patterns that allowed her to calculate
o a camera, so she could make more detailed the dimensions o the DNA helix.
measurements o the X-ray diraction patterns
than had previously been possible. She also Without Franklins knowledge or permission,
produced high quality samples o DNA with the James Watson was shown the best diraction
molecules aligned in narrow bres. By careul pattern and the calculations based on it. Beore
control o humidity two types o pure sample Franklin could publish her results Crick and
could be produced and as Franklin was unsure Watson had used them to build their model o
which represented the normal structure o DNA, DNA structure. It is widely accepted that Rosalind
she investigated both. Franklin deserved a Nobel Prize or her research,
but this never happened. C rick and Watson were
Soon ater starting work at Kings College, awarded prizes in 1 962, but she died o cancer in
Franklin had obtained the sharpest X-ray 1 958, aged thirty-seven. Nobel Prizes cannot be
diraction images o DNA in existence. They awarded posthumously, but Rosalind Franklin
have been described as amongst the most is remembered more than many prize winners.
beautiul X-ray photographs o any substance What we can remember rom her lie is that
ever taken. Their implications are described in discoveries may sometimes be made through
the next section. She was unwilling to publish serendipity or fashes o insight, but the real
her ndings until there was strong evidence. She oundations o science are rigorous experimental
thereore embarked on a rigorous analysis o the techniques and diligent observation.
Rosalind Franklins investigation of DNA structure
Rosalind Franklin and Maurice Wilkins investigation oDNA structure by X-ray difraction.
I a beam o X-rays is directed at a material,
most o it passes through but some is scattered
by the particles in the material. This scattering
is called diraction. The wavelength o X-rays
makes them particularly sensitive to diraction
by the particles in biological molecules
including DNA.
In a crystal the particles are arranged in a regular Figure 4 Rosalind Franklins X-ray difraction photograph o DNA
repeating pattern, so the diraction occurs in a
regular way. D NA cannot be crystallized but the From the diraction pattern in gure 4 Franklin
molecules were arranged in an orderly enough was able to make a series o deductions about the
array in Franklins samples or a diraction structure o DNA:
pattern to be obtained, rather than random
scattering. The cross in the centre o the pattern indicated
that the molecule was helical in shape.
An X-ray detector is placed close to the sample
to collect the scattered rays. The sample can be The angle o the cross shape showed the pitch
rotated in three dierent dimensions to investigate (steepness o angle) o the helix.
the pattern o scattering. Diraction patterns can
be recorded using X-ray lm. Franklin developed
a high resolution camera containing X-ray lm
to obtain very clear images o diraction patterns
rom DNA. Figure 4 shows the most amous o
these diraction patterns.
346
7.1 D N A s tr u ctu re AN D re pli c Ati o N
The distance between the horizontal bars the repeats. This turned out to be the vertical
showed turns o the helix to be 3.4 nm apart. distance between adjacent base pairs in the
helix.
The distance between the middle o the
diraction pattern and the top showed that These deductions that were made rom the X-ray
there was a repeating structure within the diraction pattern o DNA were critically important
molecule, with a distance o 0.34 nm between in the discovery o the structure o DNA.
The Watson and Crick model suggested semi- toK
conservative replication
Wha n d n hav
DNA structure suggested a mechanism or DNA replication. whn h and dn
dn fy mah xmna
Several lines o experimental evidence came together to lead to the vdn?
knowledge o the structure o DNA: molecular modelling pioneered
by the Nobel prize winner Linus Pauling, X-ray diraction patterns Chargaf wrote about his
discerned rom the careul photographs o Rosalind Franklin and the observations:
base composition studies o Erwin Charga. But insight and imagination
played a role as well. the results serve to disprove the
tetranucleotide hypothesis. It is,
O ne o Watson and C ricks frst models had the sugar- phosphate strands however, noteworthy - whether
wrapped around one another with the nitrogen bases acing outwards. this is more than accidental,
Rosalind Franklin countered this model with the knowledge that the cannot yet be said - that in all
nitrogen bases were relatively hydrophobic in comparison to the sugar- deoxypentose nucleic acids
phosphate backbone and would likely point in to the centre o the helix. examined thus ar the molar ratios
ototal purines to total pyrimidines
Franklins X-ray diraction studies showed that the DNA helix was and also oadenine to thymine and
tightly packed so when Watson and C rick built their models, their choices oguanine to cytosine were not ar
required the bases to ft together such that the strands were not too ar rom 1
apart. As they trialled various models, Watson and C rick ound the tight
packing they were looking or would occur i a pyrimidine was paired H. H. Bauer, author othe book
with a purine and i the bases were upside down in relation to one Scientifc Literacy and the Myth o
another. In addition to being structurally similar, adenine has a surplus the Scientifc Method, argues that
negative charge and thymine has a surplus positive charge so that pairing Chargaf needed to:
was electrically compatible. Pairing cytosine with guanine allows or the
ormation o three hydrogen bonds which enhances stability. stick his neck out beyond the
actual results and say that
Once the model was proposed, the complementary base pairing they mean exact equality and
immediately suggested a mechanism by which DNA replication could hence some sort opairing in the
occur one o the key requirements that any structural model would molecular structure . Watson
have to address. The WatsonC rick model led to the hypothesis o semi- and Crick, on the other hand
conservative replication. were speculating and theorizing
about the molecular nature and
The role of nucleosomes in DNA packing biological unctions oDNA and
they postulated a structure in
Nucleosomes help to supercoil DNA. which the equalities are exactly
one and the deviation orm this
One dierence between eukaryotic DNA and bacterial DNA is that in the data could be regarded as
eukaryotic DNA is associated with proteins called histones. Most groups o experimental error. Ideas and
prokaryotes have DNA that is not associated with histones, or proteins like theory turned out to be a better
histones. For this reason, prokaryotic DNA is reerred to as being naked. guide than raw data.
Histones are used by the cell to package the DNA into structures called 347
nucleosomes. A nucleosome consists o a central core o eight histone
7 NUCLEIC ACIDS (AHL)
H1 histone proteins with DNA coiled around the proteins. The eight proteins, or
octamer, consist o two copies o our dierent types o histones. A
DNA nucleosome short section o linker DNA connects one nucleosome to the next. An
additional histone protein molecule, called H1 , serves to bind the DNA to
Figure 5 30nm the core particle (fgure 5) .
bre
The association o histones with the DNA contributes to a pattern
known as supercoiling. An analogy is i you twist an elastic band
repeatedly eventually it orms an additional pattern o coils.
Supercoiling allows a great length o DNA to be packed into a much
smaller space within the nucleus. The nucleosome is an adaptation that
acilitates the packing o the large genomes that eukaryotes possess.
The H1 histone binds in such a way to orm a structure called the
30 nm fbre that acilitates urther packing.
Activity Visualizing nucleosomes
Determining packing ratio Utilization o molecular visualization sotware to
analyse the association between protein and DNA within
Packing ratio is defned as a nucleosome.
the length oDNA divided
by the length into which Visit the protein data bank at http://www.rcsb.org/pdb/home/home.do
it is packaged. Use the or download the image o a nucleosome rom the companion website
inormation below to estimate or this textbook.
the packing ratio o: 1 Rotate the molecule to see the two copies o each histone protein.
(a) a nucleosome; and In fgure 6, they are identifed by the tails that extend rom the
core. Each protein has such a tail that extends out rom the core.
(b) chromosome 22 (one 2 Note also the approximately 1 50 bp o DNA wrapped nearly twice
o the smallest human around the octamer core.
chromosomes) . 3 Note the N-terminal tail that projects rom the histone core or
each protein. Chemical modifcation o this tail is involved in
The distance between regulating gene expression.
base pairs is 0.34 nm. 4 Visualize the positively charged amino acids on the nucleosome
core. Suggest how they play a role in the association o the protein
There is approximately core with the negatively charged DNA.
200 bp o DNA coiled
around a nucleosome. Figure 6
A nucleosome is
approximately 10 nm
long.
There is an estimated
5.0 107 total base
pairs (bp) present in the
shortest human autosome
(chromosome 22).
Chromosome 22 in its
most condensed orm is
approximately 2 m long.
348
7.1 D N A s tr u ctu re AN D re pli c Ati o N
Daa-bad qn: Apoptosis and the length of DNA between nucleosomes
Under natural conditions, programmed cell Origin
death sometimes occurs. This is known as
apoptosis and it plays an important role in such 2000 bp
processes as metamorphosis and embryological 1500 bp
development. One mechanism involved in this
auto-destruction is the digestion o DNA by 1000 bp
enzymes called DNAases. The DNA associated 750 bp
with the nucleosome is normally not as accessible
to the DNAase as the linking sections. DNA 500 bp
gets digested into ragments o lengths equal to
multiples o the distance between nucleosomes.
The let hand column o fgure 7 shows the 250 bp
results o separation by gel electrophoresis o the
DNA released by the action o DNAase on rat Figure 7
liver cells. The right column represents ragments (iii) the length o DNA between two linker
used as a reerence called a ladder.
DNA regions with three nucleosomes
Once the DNA had been cut, nucleosomes were between them.
digested by protease. 2 Deduce the length o DNA associated with
a nucleosome.
1 Identiy on the diagram the ragment that 3 Suggest how the pattern in the let-
represents: hand column would change i very high
concentrations o DNAase were applied to
(i) the length o DNA between the two the cells.
sections o linker DNA on either side o
one nucleosome;
(ii) the length o DNA between two linker
DNA regions with two nucleosomes
between them;
The leading strand and the lagging strand
DNA replication is continuous on the leading strand and
discontinuous on the lagging strand.
Because the two strands o the DNA double helix are arranged in an
anti-parallel ashion, synthesis on the two strands occurs in very dierent
ways. One strand, the leading strand, is made continuously ollowing the
ork as it opens. The other strand, known as the lagging strand, is made
in ragments moving away rom the replication ork. New ragments are
created on the lagging strand as the replication ork exposes more o the
template strand. These ragments are called Okazaki ragments.
Proteins involved in replication
DNA replication is carried out by a complex system
of enzymes.
Replication involves the ormation and movement o the replication ork
and synthesis o the leading and lagging strands. Proteins are involved as
enzymes at each stage but also serve a number o other unctions.
349
7 NUCLEIC ACIDS (AHL)
The enzyme helicase unwinds the DNA at the replication ork and the
enzyme topoisomerase releases the strain that develops ahead o the
helicase. Single-stranded binding proteins keep the strands apart long
enough to allow the template strand to be copied.
S tarting replication requires an RNA primer. Note that on the lagging
strand there are a number o primers but there is just one on the leading
strand. The enzyme DNA primase creates one RNA primer on the
leading strand and many RNA primers on the lagging strand. The RNA
primer is necessary to initiate the activity o DNA polymerase.
DNA polymerase is responsible or covalently linking the
deoxyribonucleotide monophosphate to the 3 end o the growing
strand. Dierent organisms have dierent kinds o DNA polymerases,
each with dierent unctions such as proo-reading, polymerization and
removal o RNA primers once they are no longer needed.
DNA ligase connects the gaps between ragments.
DNA topoisomerase leading
strand
DNA polymerase
5 primase DNA ligase
3 RNA primer
parental DNA 3 lagging
helicase DNA polymerase 5 strand
DNA
Figure 8
The direction of replication
DNA polymerases can only add nucleotides to the 3 end
of a primer
Within DNA molecules, DNA replication begins at sites called origins o
replication. In prokaryotes there is one site and in eukaryotes there are
many. Replication occurs in both directions away rom the origin. The
result appears as a replication bubble in electron micrographs.
The fve carbons o the deoxyribose sugar have a number (see fgure 9) .
phosphate DNA
growing
5 O H 1 nitrogen base strand T OH C 3 end
CH2 deoxyribose sugar A C AC 5 end
H template
4 H 2 strand OH G TG G
base
H H DNA sugar phosphate
3
OH
Figure 9 Figure 10
350
7.1 D N A s tr u ctu re AN D re pli c Ati o N
The phosphate group o new DNA nucleotides is added to the the toK
3 carbon o the deoxyribose o the nucleotide at the end o the chain.
Replication thereore occurs in the 5 to 3 direction. t wha xn d n hav
a nq nby whn a
Non-coding regions o DNA have important dmay?
unctions Molecular biologist Elizabeth Blackburn
is one othe most renowned original
Some regions o DNA do not code or proteins but have researchers in the feld otelomeres.
other important unctions. She shared the Nobel Prize in Physiology
or medicine or her co-discovery o
The cellular machinery operates according to a genetic code. DNA is telomerase. She made headlines in
used as a guide or the production o polypeptides using the genetic 2004 when she was dismissed rom the
code. However, only some D NA sequences code or the production o Presidents Council on Bioethics ater
polypeptides. These are called coding sequences. There are a number o objecting to the councils call or a ban
non-coding sequences ound in genomes. Some o them have unctions, on stem cell research and or criticizing
such as those sequences that are used as a guide to produce tRNA and the suppression orelevant scientifc
rRNA. Some non-coding regions play a role in the regulation o gene evidence in its fnal report.
expression such as enhancers and silencers. In sub-topic 7.2 we will
explore non-coding sequences called introns. Figure 11 False colour scanning electron
micrograph with telomeres coloured pink. The
Most o the eukaryotic genome is non-coding. grey region in the centre is the centromere which
also consists of non-coding repetitive sequences
Within the genome, especially in eukaryotes, repetitive sequences can
be common. There are two types o repetitive sequences: moderately
repetitive sequences and highly repetitive sequences (satellite DNA) .
Together they can orm between 5 and 60 per cent o the genome. In
humans, nearly 60% o the DNA consists o repetitive sequences.
One such area o repetitive sequences occurs on the ends o eukaryotic
chromosomes called telomeres. The telomere serves a protective
unction. During interphase, the enzymes that replicate DNA cannot
continue replication all the way to the end o the chromosome. I
cells went through the cell cycle without telomeres, they would lose
the genes at the end o the chromosomes. Sacrifcing the repetitive
sequences ound in telomeres serves a protective unction.
DNA profling
Tandem repeats are used in DNA profling.
A variable number tandem repeat (VNTR) o the sequence AT and locus B has a VNTR o
is a short nucleotide sequence that shows the sequence TCG. In the two individuals shown,
variations between individuals in terms o the there are two dierent alleles (varieties) o locus A,
number o times the sequence is repeated. two repeats (allele A2) and our repeats (allele A4) .
Each variety can be inherited as an allele. In the same individuals, there are three alleles or
Analysis o VNTR allele combinations in locus B, three repeats (allele B3) , our repeats (allele
individuals is the basis behind DNA profling B4) and fve repeats (allele B5) . The asterisk mark
or use in such applications as genealogical indicates where the restriction enzyme would cut.
investigations.
The DNA profle that would result is shown in
A locus is the physical location o a heritable the lower part o fgure 1 2. Note that the two
element on the chromosome. In the hypothetical individuals have some bands in common and
example shown in fgure 1 2, locus A has a VNTR some unique bands.
351
7 NUCLEIC ACIDS (AHL)
Genealogists deduce paternal lineage by analysing mitochondrial DNA variations in single
short tandem repeats rom the Y-chromosome, nucleotides at specifc locations called hyper-
and deduce maternal lineage by analysing variable regions.
individual #1 individual #2
locus A AT AT locus A AT AT AT AT
allele A2 (2 repeats) allele A4 (4 repeats)
allele A2 (2 repeats) AT AT allele A2 (2 repeats) AT AT
locus B TCG TCG TCG locus B TCG TCG TCG
allele B3 (3 repeats) TCG TCG TCG TCG allele B3 (3 repeats)
allele B4 (4 repeats) allele B5 (5 repeats) TCG TCG TCG TCG TCG
origin DNA prole
B4
B5
B3 B3
A4
A2
individual #1 A2
individual #2
Figure 12
Activity A logarithm is an alternative way to express an exponent.
For example,
Analysis o a DNA profle involving alleles o short
tandem repeats o DNA log 1,000 = log 103 log 100 = log 102
Figure 13 Gel electrophoresis. The outside columns =3 =2
represent ladders of known length. The two inside columns
represent samples of unknown length In biology, very large changes in a variable are easier to
represent graphically i logarithms are used.
In the example (fgure 13), DNA ragments were separated
using gel electrophoresis. The ragments vary in size
rom 100 bp (base pairs) up to 5,000 bp. The two outside
columns othe gel represent ladders, i.e. mixtures oDNA
ragments oknown size. These were used to obtain the
data in table 1 and create the plot shown in fgure 14.
The other inner columns shown in fgure 13 are
unknowns.
352
7.1 D N A s tr u ctu re AN D re pli c Ati o N
Knwn add fagmn Dan mvd base pairs 104
z (b) (mm) 103
5,000 58 102
2,000 96
850 150
400 200
10 0 250
Table 1
1 Using fgure 1 4 determine the size o DNA 101
ragments in the two centre digests: 50 100 150 200 250
distance /mm
Fagmn Dan Fagmn Dan
z (b) mvd (mm) z (b) mvd (mm) Figure 14 Distance moved as a function of fragment size in
(mn 2) (mn 2) (mn 3) (mn 3) gel electrophoresis. Notice that the y-axis scale on this graph
goes up in powers of 10. This is a logarithmic scale
60 70
70 160
130 200
Daa-bad qn: Analysis o DNA profles using D1S80
One commonly studied DNA locus is a VNTR
named D1 S80. D1 S80 is located on human
chromosome 1 . This locus is composed
o repeating units o 1 6-nucleotide-long
segments o DNA. The number o repeats
varies rom one individual to the next with 29
known alleles ranging rom 1 5 repeats
to 41 .
In the image o a DNA profle (fgure 1 5) the Figure 15
outside and inside lanes represent ladders
representing multiples o one hundred and
twenty-three bp.
a) Identiy the lengths o the ragments d) Using the standard curve, estimate the
represented by each o the bands in the lengths o the bands in each individual.
la d d e r.
e) Estimate the number o repeats represented
b) Using a ruler measure the distance between by each band.
the origin and the band. Use the length and
distance data, to create a standard curve using f) It is unclear whether the individual in lane 7
a logarithmic graph. has two dierent copies o the same allele or
dierent alleles. Suggest what could be done
c) Measure the distance travelled by each band to urther resolve the genotype o the fnal
rom the origin. individual.
353
7 NUCLEIC ACIDS (AHL)
DNA sequencing
Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication
in preparation of samples for base sequencing.
The determination o the sequence o bases in a markers are added. The dideoxyribonucleotides
genome is carried out most commonly using a will be incorporated into some o the new DNA,
method that employs fuoresence. Many copies but when they are incorporated, they will stop
o the unknown DNA that is to be sequenced are the replication at precisely the point where they
placed into test tubes with all o the raw materials were added. The ragments are separated by
including deoxyribonucleotides and the enzymes length using electrophoresis. The sequence o
necessary to carry out replication. In addition bases can be automatically analysed by comparing
very small quantities o dideoxyribonucleotides the colour o the fuorescence with the length o
that have been labelled with dierent fuorescent the ragment.
DNA to be sequenced A TA G C
GA C C A
?????????
123456789 G
TA T
C mixture of nucleotides
containing rare
primer extension reactions: dideoxyribonucleotides (ddn)
ddA reaction: ddC reaction: replication stops when
a ddn is incorporated
TAC TATG C C AG A TAC TAT G C C AG A
primer ATGA AT G ATAC
ddG reaction: ddT reaction:
TAC TATG C C AG A TAC TAT G C C AG A
ATG ATAC G AT G AT
electropherogram column electrophoresis
TA C T A TG A ddn that is on the
G end of the fragment
A
C
C
G
T
A
T
C
A
T
to computer detector
laser
Figure 16
354
7. 2 tr AN s cr i pti o N AN D g e N e e Xpr e ss i o N
7.2 tann and n xn
Understanding Applications
Gene expression is regulated by proteins that The promoter as an example o non-coding DNA
bind to specifc base sequences in DNA. with a unction.
The environment o a cell and o an organism Skills
has an impact on gene expression.
Analysis o changes in DNA methylation patterns.
Nucleosomes help to regulate transcription in
e u ka ry o te s . Nature of science
Transcription occurs in a 5' to 3' direction. Looking or patterns, trends and discrepancies:
there is mounting evidence that the
Eukaryotic cells modiy mRNA ater environment can trigger heritable changes in
transcription. epigenetic actors.
Splicing o mRNA increases the number o
dierent proteins an organism can produce.
The function of the promoter
The promoter as an example o non-coding DNA with a unction.
Only some DNA sequences code or the production The promoter is a sequence that is located
o polypeptides. These are called coding sequences. near a gene. It is the binding site o RNA
There are a number o non-coding sequences ound polymerase, the enzyme that catalyses the
in genomes. Some o them have unctions, such as ormation o the covalent bond between
those sequences that produce tRNA and rRNA. nucleotides during the synthesis o RNA. The
promoter is not transcribed but plays a role
Some non-coding regions play a role in the regulation in transcription.
o gene expression such as enhancers and silencers.
Regulation of gene expression by proteins 355
Gene expression is regulated by proteins that bind to
specifc base sequences in DNA.
Some proteins are always necessary or the survival o the organism
and are thereore expressed in an unregulated ashion. Other proteins
need to be produced at certain times and in certain amounts; i.e., their
expression must be regulated.
Gene expression is regulated in prokaryotes as a consequence o
variations in environmental actors. For example, the genes responsible
or the absorption and metabolism o lactose by E.coli are expressed in
the presence o lactose and are not expressed in the absence o lactose.
In this case, the breakdown o lactose results in regulation o gene
expression by negative eedback. In the presence o lactose a repressor
protein is deactivated (fgure 1 ) . Once the lactose has been broken
7 NUCLEIC ACIDS (AHL)
lactose not in the environment; down, the repressor protein is no longer deactivated and proceeds to
repressor blocks transcription block the expression o lactose metabolism genes.
prom oter As in prokaryotes, eukaryotic genes are regulated in response to
variations in environmental conditions. Each cell o a multicellular
lactose present in the environment; eukaryotic organism expresses only a raction o its genes.
repressor deactivated; genes involved in lactose
use are transcribed The regulation o eukaryotic gene expression is also a critical part o
cellular dierentiation as well as the process o development. This is
promoter seen in the passage o an insect through its lie cycle stages or in human
embryological development.
RNA polymerase
There are a number o proteins whose binding to DNA regulates
-galactosidase transacetylase transcription. These include enhancers, silencers and promoter-proximal
elements. Unlike the promoter sequence, the sequences linked to
-+ permease regulatory transcription actors are unique to the gene.
- Regulatory sequences on the DNA which increase the rate o
transcription when proteins bind to them are called enhancers. Those
- sequences on the DNA which decrease the rate o transcription when
- proteins bind to them are called silencers. While enhancers and silencers
can be distant rom the promoter, another series o sequences called
- lactose promoter-proximal elements are nearer to the promoter and binding
o proteins to them is also necessary to initiate transcription.
Figure 1
The impact ofthe environment on gene expression
The environment of a cell and of an organism has an
impact on gene expression.
In the history o Western thought, much debate has gone in to the
naturenurture debate. This is a debate centred on the extent to which
a particular human behaviour or phenotype should be attributed to
the environment or to heredity. Much eort has gone into twin studies
especially or twins raised apart.
Data-based questions: Identical twin studies percent of twin pairs who share the trait
Twin studies have been used to identiy 0% 100%
the relative infuence o genetic actors and height greater
environmental actors in the onset o disease genetic
(gure 2) . Identical twins have 1 00% o the same inuence
DNA while raternal twins have approximately reading disability
50% o the same DNA.
autism
Questions
Alzheimers
schizophrenia
alcoholism
1 Determine the percentage o identical twins bipolar disorder
where both have diabetes. [2] hypertension
2 Explain why a higher percentage o diabetes
identical twins sharing a trait suggests that multiple sclerosis
a genetic component contributes to the breast cancer
onset o the trait. [3] Crohns disease
3 With reerence to any our conditions, discuss stroke identical twins greater
rheumatoid arthritis environmental
the relative role o the environment and fraternal twins inuence
Figure 2
genetics in the onset o the condition. [3]
356
7. 2 tr AN s cr i pti o N AN D g e N e e Xpr e ss i o N
The infuence o the environment on gene expression or some traits Avy
is unequivocal. Environmental actors can aect gene expression such
as the production o skin pigmentation during exposure to sunlight exlan h a lu
in humans. an f sam a
In embryonic development, the embryo contains an uneven distribution
o chemicals called morphogens. Concentrations o the morphogens
aect gene expression contributing to dierent patterns o gene
expression and thus dierent ates o the embryonic cells depending on
their position in the embryo.
In coat colour in cats, the C gene codes or the production o the
enzyme tyrosinase, the rst step in the production o pigment. A
mutant allele o the gene, cs allows normal pigment production
only at temperatures below body temperature. This mutant allele has
been selected or in the selective breeding o Siamese cats. At higher
temperatures, the protein product is inactive or less active, resulting in
less pigment.
Nucleosomes regulate transcription
Nucleosomes help to regulate transcription in eukaryotes.
Eukaryotic DNA is associated with proteins called histones. Chemical
modication o the tails o histones is an important actor in determining
whether a gene will be expressed or not.
A number o dierent types o modication can occur to the tails o
histones including the addition o an acetyl group, the addition o a
methyl group or the addition o a phosphate group.
O Acetyl group CH3- Methyl group
CH3C -
For example, residues o the amino acid lysine on histone tails can have MM
acetyl groups either removed or added. Normally the lysine residues GCTGA C T G
on histone tails bear a positive charge that can bind to the negatively
charged DNA to orm a condensed structure that inhibits transcription.
Histone acetylation neutralizes these positive charges allowing a less
condensed structure with higher levels o transcription.
Chemical modication o histone tails can either activate or deactivate CGAC T G A C
genes by decreasing or increasing the accessibility o the gene to
transcription actors. MM M
NH2
Analysing methylation patterns C CH3
NC
Analysis of changes in DNA methylation patterns
CC
The addition o methyl groups directly to DNA is thought to play a ON
role in gene expression. Whereas methylation o histones can promote
or inhibit transcription, direct methylation o DNA tends to decrease Figure 3 DNA methylation is the addition
gene expression. The amount o DNA methylation varies during a of a methyl group (green M) to the DNA
lietime and is aected by environmental actors. base cytosine
357
7 NUCLEIC ACIDS (AHL)
Data-basd qustions: Changes in methylation pattern with age in identical twins
One study compared the methylation patterns each other in their characteristics as they [2]
o 3-year-old identical twins with 50-year-old grow older.
identical twins. Methylation patterns were dyed
red on one chromosome or one twin and dyed
green or the other twin on the same chromosome.
Chromosome pairs in each set o twins were digitally
superimposed. The result would be a yellow colour
i the patterns were the same. Dierences in patterns
on the two chromosomes results in mixed patterns
o green and red patches. This was done or our o
the twenty-three chromosome pairs in the genome.
1 Explain the reason or yellow coloration
i the methylation pattern is the same
in the two twins. [3]
2 Identiy the chromosome with the least
changes as twins age. [1 ]
3 Identiy the chromosomes with the most
changes as twins age. [1 ]
4 Explain how these dierences could arise. [3]
5 Predict with a reason whether identical Figure 4
twins will become more or less similar to
epigntics
Looking for patterns, trends and discrepancies: there is mounting evidence that
the environment can trigger heritable changes in epigenetic factors.
The chemical modifcations o chromatin that Sperm and eggs develop rom cells with
impact gene expression, including acetylation, epigenetic tags. When two reproductive cells
methylation and phosphorylation o amino meet, the epigenome is erased through a process
acid tails o histones (fgure 5) as well as called reprogramming.
methylation o DNA(fgure 6) , all have an impact
on gene expression and thus impact the visible Ac acetylation
characteristics o an individual (fgure 7) . These M methylation
chemical modifcations are called epigenetic tags. P phosphorylation
There is mounting evidence that the chemical
modifcations that occur to the hereditary material Figure 5 Histone modifcations
in one generation might, in certain circumstances,
be passed on to the next generation both at the NH2 NH2
cellular as well as whole organism level. The sum
o all the epigenetic tags constitutes the epigenome.
Dierent cells have their own methylation pattern CH C Me
so that a unique set o proteins will be produced NC NC
in order or that cell to perorm its unction.
During cell division, the methylation pattern C CH C CH
will be passed over to the daughter cell. In other ON ON
words, the environment is aecting inheritance.
H H
Figure 6 DNA methylation
358
7. 2 tr AN s cr i pti o N AN D g e N e e Xpr e ss i o N
About 1 % o the epigenome is not erased and the etal circulation trigger epigenetic changes in
survives yielding a result called imprinting. the daughters DNA such that she is predisposed
to develop gestational diabetes hersel.
For example, when a mammalian mother has
gestational diabetes, the high levels o glucose in transcription possible
gene switched on
active (open) chromatin
unmethylated cytosines
(white circles)
acetylated histones
gene switched o
silent (condensed) chromatin
methylated cytosines
(red circles)
deacetylated histones
transcription prevented
Figure 7 The diagram compares the chemical modifcations that prevent transcription with the
chemical modifcations that allow transcription
The direction o transcription
Transcription occurs in a 5' to 3' direction.
The synthesis o mRNA occurs in three stages: initiation, elongation
and termination. Transcription begins near a site in the D NA called the
promoter. O nce binding o the RNA polymerase occurs, the D NA is
unwound by the RNA polymerase orming an open complex. The RNA
polymerase slides along the DNA, synthesizing a single strand o RNA.
RNA base
growing
OH 3 end
5 end
strand OH OH OH OH
U C AC
A G TG G C
template
strand OH
DNA sugar phosphate
Figure 8
Post-transcriptional modifcation Figure 9 Coloured transmission electron
micrograph o DNA transcription coupled
Eukaryotic cells modify mRNA after transcription. with translation in the bacterium Escherichia
coli. During transcription, complementary
The regulation o gene expression can occur at several points. Transcription, messenger ribonucleic acid (mRNA) strands
translation and post-translational regulation occur in both eukaryotes and (green) are synthesized using DNA (pink)
prokaryotes. However, most regulation o prokaryotic gene expression as a template and immediately translated by
occurs at transcription. In addition, post-transcriptional modifcation o RNA ribosomes (blue)
is a method o gene expression that does not occur in prokaryotes.
359
7 NUCLEIC ACIDS (AHL)
a) O
HH
N
H2N HH
N N O OOO O base
H
OH HO CH2 O P O P O P O CH2
O
H OOO HH
HH
N O OH
CH3 O P O CH2 3
O 5
7-methylguanosine cap
b) exon intron exon
5 pre-mRNA 3
toK spliceosome sn RNPs
Hw d he crieria fr exon exon
judgmen used change he 5 3
cnclusins drawn frm he
same daa? excised 3
intron
Estimates othe number o A poly A tail consisting of
genes ound in the human 5 100200 adenine nucleotides
genome fuctuated wildly in mature mRNA is added after transcription.
the time between 2000 and A A A A A 3
2007. Reported as high as c)
120,000 in 2000, the current
consensus view is that there 5
are approximately 20,500.
The reason or the uncertainty poly A tail
was due to the dierent
criteria used or searching Figure 10
used by dierent gene-nding
programs. One o the most signifcant dierences between eukaryotes and
prokaryotes is the absence o a nuclear membrane surrounding the
Dening the criteria was genetic material in prokaryotes. The absence o a compartment in
problematic because: prokaryotes means that transcription and translation can be coupled.
small genes are diicult The separation o the location o transcription and translation into separate
to detect; compartments in eukaryotes allows or signifcant post-transcriptional
modifcation to occur beore the mature transcript exits the nucleus. An
because o mRNA example would be the removal o intervening sequences, or introns, rom
splicing, one gene can the RNA transcript. Prokaryotic DNA does not contain introns.
code or several protein
products; In eukaryotes, the immediate product o mRNA transcription is
reerred to as pre-mRNA, as it must go through several stages o post-
some genes are non- transcriptional modifcation to become mature mRNA.
protein coding and two
genes can overlap. One o these stages is called RNA splicing, shown in fgure 1 1 b.
Interspersed throughout the mRNA are sequences that will not
360
7. 2 tr AN s cr i pti o N AN D g e N e e Xpr e ss i o N
contribute to the ormation o the polypeptide. They are reerred to as
intervening sequences, or introns. These introns must be removed. The
remaining coding portions o the mRNA are called exons. These will be
spliced together to orm the mature mRNA.
Post-transcriptional modication also includes the addition o a 5 cap that
usually occurs beore transcription has been completed (see gure 1 1 a) .
A poly-A tail is added ater the transcript has been made (see gure 1 1 c) .
mRNA splicing
Splicing o mRNA increases the number o diferent
proteins an organism can produce.
Alternative splicing is a process during gene expression whereby a single
gene codes or multiple proteins. This occurs in genes with multiple
exons. A particular exon may or may not be included in the nal
messenger RNA. As a result, the proteins translated rom alternatively
spliced mRNAs will dier in their amino acid sequence and possibly in
their biological unctions.
In mammals, the protein tropomyosin is encoded by a gene that has
eleven exons. Tropomyosin pre- mRNA is spliced dierently in dierent
tissues resulting in ve dierent orms o the protein. For example, in
skeletal muscle, exon 2 is missing rom the mRNA and in smooth
muscle, exons 3 and 1 0 are not present.
In ruit fies, the Dscam protein is involved in guiding growing nerve cells
to their targets. Research has shown that there are potentially 38,000
dierent mRNAs possible based on the number o dierent introns in the
gene that could be spliced alternatively.
361
7 NUCLEIC ACIDS (AHL)
7.3 translaion
Understanding Applications
Initiation o translation involves assembly o tRNA-activating enzymes illustrate enzyme-
the components that carry out the process. substrate specifcity and the role o
phosphorylation.
Synthesis o the polypeptide involves a
repeated cycle o events. Skills
Disassembly o the components ollows The use o molecular visualization sotware to
termination o translation. analyse the structure o eukaryotic ribosomes
and a tRNA molecule.
Free ribosomes synthesize proteins or use
primarily within the cell. Identifcation o polysomes in an electron
micrograph.
Bound ribosomes synthesize proteins primarily
or secretion or or use in lysosomes. Nature of science
Translation can occur immediately ater Developments in scientifc research ollow
transcription in prokaryotes due to the absence improvements in computing: the use o
o a nuclear membrane. computers has enabled scientists to make
advances in bioinormatics applications
The sequence and number o amino acids in such as locating genes within genomes and
the polypeptide is the primary structure. identiying conserved sequences.
The secondary structure is the ormation
o alpha helices and beta pleated sheets
stabilized by hydrogen bonding.
The tertiary structure is the urther olding
o the polypeptide stabilized by interactions
between R groups.
The quaternary structure exists in proteins with
more than one polypeptide chain.
The structure of the ribosome
The use o molecular visualization sotware to analyse the structure o eukaryotic
ribosomes and a tRNA molecule.
Ribosome structure includes: Each ribosome has three tRNA binding sites the
E or exit site, the P or peptidyl site and the
Proteins and ribosomal RNA molecules (rRNA) . A or aminoacyl site (see fgure 1 ) .
Two sub-units, one large and one small. The protein data bank (PDB) is a public
database containing data regarding the three-
Three binding sites or tRNA on the surace o dimensional structure or a large number o
the ribosome. Two tRNA molecules can bind at biological molecules. In 2000, structural biologists
the same time to the ribosome. Venkatraman Ramakrishnan, Thomas A. S teitz
and Ada E . Yonath made the frst data about
There is a binding site or mRNA on the
surace o the ribosome.
362
7. 3 trAN s l Ati o N
position of large tRNA structure A 3 site for attaching
growing polypeptide sub-unit C an amino acid
double stranded sections C
binding sites small linked by base pairing 5 loop of seven
for tRNA sub-unit nucleotides
5 loop of eight extra loop
3 nucleotides anticodon loop
position of mRNA
Figure 1
ribosome subunits available through the PDB. In anticodon
2009, they received a Nobel Prize or their work
on the structure o ribosomes. Figure 3
Visit the protein databank to obtain images a triplet o bases called the anticodon which is
o the Thermus thermophilus ribosome ( images part o a loop o seven unpaired bases
1 jgo and 1 giy) , or download these images rom
the companion website to the textbook. Using two other loops
Jmol, rotate the image to visualize the small
sub-unit and the large sub-unit. In the image in the base sequence CCA at the 3' end which
fgure 2 , an mRNA molecule is coloured yellow. orms a site or attaching an amino acid.
The pink, purple and blue areas in the image
represent the three tRNA binding sites with tRNA Visit the PD B to obtain an image o a tRNA
molecules bound. molecule or download the image rom the
companion site to this book to explore the
structure in a programme such as Jmol. Figure 4
shows such an image. The parts marked green
represent the amino acid binding site and the anti-
codon. The part in purple shows a region o the
molecule where a triplet o bases are hydrogen
bonded. This is shown in the second image.
Figure 2
The generalized structure o a tRNA molecule is Figure 4 Whole view of a tRNA molecule with a close-up of a
shown in fgure 3. triplet of bases connected by hydrogen bonds
All tRNA molecules have:
sections that become double-stranded by base
pairing, creating loops
363
7 NUCLEIC ACIDS (AHL)
tRNA-activating enzymes
tRNA-activating enzymes illustrate enzyme-substrate specifcity and the role
o phosphorylation.
Each tRNA molecule is recognized by a tRNA- activating enzyme is specifc to both the correct
activating enzyme that attaches a specifc amino amino acid and the correct tRNA.
acid to the tRNA, using ATP or energy.
Energy rom ATP is needed or the attachment
The base sequence o tRNA molecules varies and o amino acids. Once ATP and an amino acid are
this causes some variability in structure. Activation attached to the active site o the enzyme, the amino
o a tRNA molecule involves the attachment o an acid is activated by the ormation o a bond between
amino acid to the 3' terminal o the tRNA by an the enzyme and adenosine monophosphate (AMP) .
enzyme called a tRNA-activating enzyme. There Then the activated amino acid is covalently attached
are twenty dierent tRNA-activating enzymes that to the tRNA. Energy rom this bond is later used
are each specifc to one o the 20 amino acids and to link the amino acid to the growing polypeptide
the correct tRNA molecule. The active site o the chain during translation.
ATP tRNA
PPP charged tRNA
amino acid
P
aminoacyl-tRNA P Pi P The activated
synthetase pyrophosphate tRNA is released
AMP
A specic amino acid The amino acid is a
and ATP bind to the activated by the The correct tRNA binds to
enzyme hydrolysis of ATP and the active site. The amino
covalent bonding acid binds to the attachment
Figure 5 of AMP site on the tRNA and AMP is
released
3 U A C 5 Initiation of translation
Met 5 A U G 3
initiator tRNA Initiation o translation involves assembly o the
components that carry out the process.
5 3
start codon To begin the process o translation, an mRNA molecule binds to
small the small ribosomal subunit at an mRNA binding site. An initiator
mRNA binding site ribosomal tRNA molecule carrying methionine then binds at the start codon
subunit AUG.
Figure 6
The large ribosomal subunit then binds to the small one.
The initiator tRNA is in the P site. The next codon signals another tRNA
to bind. It occupies the A site. A peptide bond is ormed between the
amino acids in the P and A site.
364
7. 3 trAN s l Ati o N
l a rge peptide bond
forming
P site Met ribosomal
subunit E
PA
E A E
5
3
Figure 7
3 PA
site site
5
Figure 8
Elongation of the polypeptide
Synthesis of the polypeptide involves a repeated cycle
of events.
Following initiation, elongation occurs through a series of repeated steps.
The ribosome translocates three bases along the mRNA, moving the
tRNA in the P site to the E site, freeing it and allowing a tRNA with the
appropriate anticodon to bind to the next codon and occupy the vacant
A site.
E E
PA 3
PA
5 site site
Figure 9
Termination of translation
Disassembly of the components follows termination of
translation.
The process continues until a stop codon is reached when the free
polypeptide is released. Note the direction of movement along the
mRNA is from the 5end to the 3 end.
free polypeptide
3 3
5 5
Figure 10 stop codon
(UAG, UAA, or UGA)
365
7 NUCLEIC ACIDS (AHL)
toK Free ribosomes
Hw d wrds acquire heir meaning? Free ribosomes synthesize proteins or use primarily
within the cell.
Is a ribosome an organelle? Karl
August Mbius is credited as the frst In eukaryotes, proteins unction in a particular cellular compartment.
to establish the analogy between Proteins are synthesized either in the cytoplasm or at the endoplasmic
cellular substructures with defned reticulum depending on the fnal destination o the protein. Translation
unctions and the organs othe body. occurs more commonly in the cytosol. Proteins destined or use in the
Early usage varied rom reerring only to cytoplasm, mitochondria and chloroplasts are synthesized by ribosomes
the reproductive structures oprotists, ree in the cytoplasm.
later ocusing on propulsion structures
and later even including extracellular Bound ribosomes
structures such as cell walls. The
original defnition oan organelle as a Bound ribosomes synthesize proteins primarily or
subcellular unctional unit in general has secretion or or use in lysosomes.
emerged as the dominant defnition, and
this would include ribosomes. A criterion In eukaryotic cells, thousands o proteins are made. In many cases,
in this case or defning an organelle is proteins perorm a unction within a specifc compartment o the cell or
whether it can be isolated by a process they are secreted. Proteins must thereore be sorted so that they end up
known as cellular ractionation. Others in their correct location. Proteins that are destined or use in the ER, the
limit the term to membrane-bound cell Golgi apparatus, lysosomes, the plasma membrane or outside the cell are
compartments and some cell biologists synthesized by ribosomes bound to the ER.
choose to limit the term even urther to
those structures that originated rom Whether the ribosome is ree in the cytosol or bound to the ER
endosymbiotic bacteria. depends on the presence o a signal sequence on the polypeptide
vesicle containing
polypeptide
ribosome
mRNA
signal
sequence
signal recognition
protein (SRP)
polypeptide ER membrane
SRP receptor
lumen of ER
Figure 11
366
7. 3 trAN s l Ati o N
being translated. It is the frst part o the polypeptide translated.
As the signal sequence is created it becomes bound to a signal
recognition protein that stops the translation until it can bind to a
receptor on the surace o the ER. Once this happens, translation
begins again with the polypeptide moving into the lumen o the ER as
it is created.
The coupling o transcription and translation in
prokaryotes
Translation can occur immediately ater transcription in
prokaryotes due to the absence o a nuclear membrane.
In eukaryotes, cellular unctions are compartmentalized whereas in
prokaryotes they are not. Once transcription is complete in eukaryotes,
the transcript is modifed in several ways beore exiting the nucleus.
Thus there is a delay between transcription and translation due
to compartmentalization. In prokaryotes, as soon as the mRNA is
transcribed, translation begins.
Identifcation opolysomes
Identifcation o polysomes in an electron micrograph.
Polysomes are structures visible in an electron compartment in prokaryotes, as soon as the
microscope. They appear as beads on a mRNA is transcribed, translation begins. Thus,
string. They represent multiple ribosomes multiple polysomes are visible associated with
attached to a single mRNA molecule. Because one gene. In eukaryotes, polysomes occur in
translation and transcription occur in the same both the cytoplasm and next to the ER.
Figure 12 Strings of polysomes attached to a DNA molecule in a prokaryote. The arrow designates where investigators believe RNA
polymerase is sitting at, or near, the initiation site for a gene
367
7 NUCLEIC ACIDS (AHL)
polypeptide
ribosome
mRNA
Figure 13 The image shows multiple ribosomes translating a single mRNA molecule within the cytoplasm at the same time.
The beginning of the mRNA is to the right (at the arrow) . The polypeptides being synthesized get longer and longer, the
closer the end of the mRNA the ribosomes get
Bioinformatics
Developments in scientifc research ollow improvements in computing: the use o
computers has enabled scientists to make advances in bioinormatics applications
such as locating genes within genomes and identiying conserved sequences.
Bioinormatics involves the use o computers to The unctions o conserved sequences are oten
store and analyse the huge amounts o data being investigated in model organisms such as E. coli,
generated by the sequencing o genomes and the yeast ( S. cerevisiae) , ruit fies ( D. melanogaster) , a
identication o gene and protein sequences. soil roundworm C. elegans, thale cress A. thalania
and mice M. musculus. These particular organisms
Such inormation is oten amassed in databases, are oten used because, along with humans, their
or example, GenBank (a US-based database) , the entire genomes have been sequenced.
DDBJ (DNA databank o Japan) or the nucleotide
sequence database maintained by the EMBL (the Functions are oten discovered by knockout studies
European Molecular Biology Laboratory) , which where the conserved gene is disrupted or altered and
then become accessible to the global community the impact on the organisms phenotype is observed.
including scientists and the general public.
A scientist studying a particular genetic disorder
in humans might identiy sequence similarities
that exist in people with the disorder. They
might then search or homologous sequences in
other organisms. These sequences might have a
common ancestral origin but have accumulated
dierences over time due to random mutation.
To carry out the search or a homologous Figure 14 Examples of model organisms
nucleotide or amino acid sequence, the scientist
would conduct a BLAST search. The acronym In addition to the BLAST program, there are
stands or basic local alignment search tool. other sotware programs available. ClustalW can
be used to align homologous sequences to search
Sometimes the homologous sequences are identical or changes. PhyloWin can be used to construct
or nearly identical across species. These are called evolutionary trees based on sequence similarities.
conserved sequences. The act that they are conserved
across species suggests they play a unctional role.
368
7. 3 trAN s l Ati o N
Primary structure
The sequence and number of amino acids in the
polypeptide is the primary structure.
A chain of amino acids is called a polypeptide. Given that the 20 commonly
occurring amino acids can be combined in any sequence, it should not be
surprising that there is a huge diversity of proteins.
The sequence of amino acids in a polypeptide is termed its primary
structure.
Daa-baed quen glu phe thr pro ala val his ala ser leu [4]
asp lys phe leu ala ser val ser thr val
The hemoglobin molecule transports oxygen in leu thr ser lys tyr arg 1 41
the blood. It consists of 4 polypeptide chains.
In human adults the molecule has two kinds of beta chain:
chains, alpha chains and beta chains, and there
are two each. The alpha chain has 1 41 amino 1 val his leu thr pro glu glu lys ser ala
acid residues and the beta chain has 1 46 amino val thr ala leu trp gly lys val asn * * val
acid residues. The primary sequence of both asp glu val gly gly glu ala leu gly arg
polypeptides is shown below. The single residue leu leu val val tyr pro trp thr gln arg
in the beta chain marked in blue is the site of a phe phe glu ser phe gly asp leu ser thr
mutation in sickle cell anemia. In the mutation, pro asp ala val met gly asn pro lys val
the glutamic acid is replaced by valine. lys ala his gly lys lys val leu gly ala phe
ser asp gly leu ala his leu asp asn leu
alpha chain: lys gly thr phe ala thr leu ser glu leu
his cys asp lys leu his val asp pro glu
1 val * leu ser pro ala asp lys thr asn asn phe arg leu leu gly asn val leu val
val lys ala ala trp gly lys val gly ala cys val leu ala his his phe gly lys glu
his ala gly glu tyr gly ala glu ala leu phe thr pro pro val gln ala ala tyr gln
glu arg met phe leu ser phe pro thr lys val val ala gly val ala asp ala leu ala
thr lys thr tyr phe pro his phe * asp his lys tyr his 1 46
leu ser his gly ser ala * * * * * gln val
lys gly his gly lys lys val ala asp ala Compare the primary structure of the
leu thr asn ala val ala his val asp asp two polypeptides. The asterix (*) symbols
met pro asn ala leu ser ala leu ser asp indicates locations where sections of
leu his ala his lys leu arg val asp pro the amino acid sequence are missing to
val asp phe lys leu leu ser his cys leu facilitate comparison.
leu val thr leu ala ala his leu pro ala
Secondary structure
The secondary structure is the formation of alpha
helices and beta pleated sheets stabilized by
hydrogen bonding.
Because the chain of amino acids in a polypeptide has polar covalent
bonds within its backbone, it tends to fold in such a way that hydrogen
bonds form between the carboxyl (C=O) group of one residue and the
amino group (NH) group of an amino acid in another part of the
chain. This results in the formation of patterns within the polypeptide
called secondary structures. The -helix and the -pleated sheet are
examples of secondary structures.
369
7 NUCLEIC ACIDS (AHL)
(a) alpha helix
CH
ON CC H
(b) beta pleated sheet
N
O C OH OH OH O
HH C C C C N C NC C N C CNC C NC
N C C
O C N
hydrogen
C N CCN bond H OH OH OH
C HO C H O CH O CH OH O
N O N CN C N CN C C N C CN C
N C HO O HO C
OC C C HO
Figure 15 The structure of insulin showing H HC
three areas where the -helix can be seen.
It also shows the quaternary structure of NCCN O
insulin, i.e. the relative positions of the two
polypeptides O
Figure 16 Two examples of protein secondary structure
Tertiary structure
The tertiary structure is the further folding of the
polypeptide stabilized by interactions between R groups.
Tertiary structure reers to the overall three- dimensional shape o the
protein (fgure 1 8) . This shape is a consequence o the interaction o
R-groups with one another and with the surrounding water medium.
There are several dierent types o interaction.
Positively charged R-groups will interact with negatively charged R-groups.
Hydrophobic amino acids will orientate themselves toward the centre
o the polypeptide to avoid contact with water, while hydrophilic
amino acids will orientate themselves outward.
Polar R-groups will orm hydrogen bonds with other polar R-groups.
The R-group o the amino acid cysteine can orm a covalent bond
with the R-group o another cysteine orming what is called a
disulphide bridge.
CH2 CH hydrophobic
interaction
O H3C CH3
H H3C CH3 polypeptide
backbone
CH
hydrogen
bond
O
C OH CH2 S S CH2
disulphide bridge
CH2
Figure 17 Collagenthe quaternary CH2 CH2 CH2 CH2 NH3 O O
structure consists of three polypeptides ionic bond C CH2
wound together to fom a tough, rope-like
protein
370 Figure 18 R-group interactions contribute to tertiary structure
7. 3 trAN s l Ati o N
Quartenary structure beta chain beta chain
The quaternary structure exists in proteins with more than alpha chain heme alpha chain
one polypeptide chain.
Figure 19 The quaternary structure of
Proteins can be ormed rom a single polypeptide chain or rom more hemoglobin in adults consists of four chains:
than one polypeptide chain. Lysozyme is composed o a single chain, two -chains and two -chains. Each subunit
so lysozyme is both a polypeptide and a protein. Insulin is ormed contains a molecule called a heme group
rom two polypeptides, and hemoglobin is made up o our chains.
Quaternary structure reers to the way polypeptides ft together
when there is more than one chain. It also reers to the addition
o non-polypeptide components. The quaternary structure o the
hemoglobin molecule consists o our polypeptide chains and our
heme groups.
The biological activity o a protein is related to its primary, secondary,
tertiary and quaternary structure. Certain treatments such as exposure
to high temperatures, or changes in pH can cause alterations in the
structure o a protein and thereore disrupt its biological activity. When a
protein has permanently lost its structure it is said to be denatured.
Daa-baed quen or the changes in hemoglobin type during
Hemoglobin is a protein composed o two pairs o development and ater birth. [3]
globin subunits. During the process o development
rom conception through to 6 months ater birth, Key
human hemoglobin changes in composition. Adult a l p h a - gl o b i n
hemoglobin consists o two alpha- and two beta- gamma-globin
globin subunits. Four other polypeptides are ound b e t a - gl o b i n
during development: zeta, delta, epsilon and gamma. d e l t a - gl o b i n
epsilon-globin
Figure 20 illustrates the changes in hemoglobin zeta-globin
composition during gestation and ater birth in a % hemoglobin
human. 50
40
a) State which two subunits are present in 30
20
highest amounts early in gestation. [1 ] 10
0
b) Compare changes in the amount o the [3]
gamma-globin gene with beta-globin.
c) Determine the composition o the
hemoglobin at 1 0 weeks o gestation and
at 6 months o age. [2]
d) State the source o oxygen or the etus. [1 ] 10 20 30 40 2 4 6
Weeks of gestation Birth Month of age
e) The dierent types o hemoglobin have
dierent afnities or oxygen. Suggest reasons Figure 20
371
37 N U C LE I C AC I D S
Questions
1 Dierent samples o bacteria were supplied 2 With reerence to Figure 22, answer the
with radioactive nucleoside triphosphates or a ollowing questions.
series o times (1 0, 30 or 60 seconds) . This was
the pulse period. This was ollowed by adding B
a large excess o non-radioactive nucleoside A
triphosphates or a longer period o time. This
is called the chase period. The appearance o C
radioactive nucleotides (incorporated during
the pulse) in parts o the product DNA give
an indication o the process o converting
intermediates to fnal products.
DNA was isolated rom the bacterial cells, D
denatured (separated into two strands by heat)
and centriuged to separate molecules by size.
The closer to the top o the centriuge tube, the
smaller the molecule.
a) Compare the sample that was pulsed or
1 0 seconds with the sample that was E
pulsed or 30 seconds. [2] Figure 22
b) Explain why the sample that was pulsed a) What part o the nucleotide is labelled A? [1 ]
or 30 seconds provides evidence or the
presence o both a leading strand and many b) What kind o bond orms between the
lagging strands.
structures labelled B? [1 ]
c) Explain why the sample that was pulsed c) What kind o bond is indicated by [1 ]
or 60 seconds provides evidence or the label C?
activity o DNA ligase.
d) What sub-unit is indicated by label D? [1 ]
Radioactivity cpm / 0.1 m1 6,000 e) What sub-unit is indicated by label E? [1 ]
60 sec
5,000 3 Reer to fgure 23 when answering the
4,000 ollowing questions.
3,000
30 sec V I
CH2OH H
O
2,000 10 sec IV H OH
1,000 HH II
OH H
III
0 0 1 23 Figure 23
Distance from top
Figure 21 a) State what molecule is represented. [1 ]
[1 ]
b) State whether the molecule would be [1 ]
ound in DNA or RNA. [1 ]
c) State the part o the molecule to which
phosphates bind.
d) Identiy the part o the molecule that
reers to the 3 end.
372
8 M ETABOLISM , CELL RESPIRATION
C E LALN DB IPOHLOOTGOYSYN TH E SI S ( AH L)
Introduction usable form in cell respiration. In photosynthesis
light energy is converted into chemical energy
Life is sustained by a complex web of chemical and a huge diversity of carbon compounds is
reactions inside cells. These metabolic reactions produced.
are regulated in response to the needs of the
cell and the organism. Energy is converted to a
8.1 Metabolism
Understanding Applications
Metabolic pathways consist o chains and End-product inhibition o the pathway that
cycles o enzyme-catalysed reactions. converts threonine to isoleucine.
Enzymes lower the activation energy o the Use o databases to identiy potential new
chemical reactions that they catalyse. anti-malarial drugs.
Enzyme inhibitors can be competitive or Skills
non-competitive.
Distinguishing diferent types o inhibition rom
Metabolic pathways can be controlled by graphs at specied substrate concentration.
end-product inhibition.
Calculating and plotting rates o reaction rom
raw experimental results.
Nature of science
Developments in scientic research ollow improvements in computing: developments in bioinormatics,
such as the interrogation o databases, have acilitated research into metabolic pathways.
373
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
TREAD initial Metabolic pathways
BREAD substrate
BREED intermediates Metabolic pathways consist of chains and cycles of
BLEED enzyme-catalysed reactions.
BLEND end product
BLIND The word metabolism was introduced in the 1 9th century by the
BLINK German cytologist and physiologist Theodor Schwann, to reer to the
chemical changes that take place in living cells. It is now known that a
Figure 1 Word game analogy for huge range o chemical reactions occur in cells, catalysed by over 5,000
metabolic pathways dierent types o enzyme. Although metabolism is very complex, there
are some common patterns.
phenylalanine
I 1 Most chemical changes happen not in one large jump, but in a
sequence o small steps, together orming what is called a metabolic
tyrosine pathway. The word game in fgure 1 is an analogy.
II
2 Most metabolic pathways involve a chain o reactions. Figure 2 shows
hydroxyphenylpyruvate a reaction chain that is used by cells to convert phenylalanine into
III umarate and acetoacetate, which can be used as energy sources in
respiration. Phenylalanine causes severe health problems i there is
homogentisate an excess o it in the blood.
IV
3 S ome metabolic pathways orm a cycle rather than a chain. In this
4-maleylacetoacetate type o pathway, the end product o one reaction is the reactant that
V starts the rest o the pathway.
4-fumarylacetoacetate input: 3 CO2 NADH + H+ acetyl group C2
VI
N AD +
fumarate + acetoacetate
RuBP 3-PGA 6 ATP FADH 2 Krebs
Figure 2 Example of a metabolic 6 G3P FAD cycle
pathway 3 ADP 6 ADP + P
3 ATP Calvin 6 NADPH C4 compound C6 compound
cycle 6 NADP+ CO2
5 G3P NADH + H+ N AD +
H+
N AD + ADH +
N
C5
compound
output: 1 G3P glucose and CO2
other compounds
Figure 3
Enzymes and activation energy
Enzymes lower the activation energy of the chemical
reactions that they catalyse.
Chemical reactions are not single-step processes. Substrates have
to pass through a transition state beore they are converted into
products. Energy is required to reach the transition state, and
although energy is released in going rom the transition state to the
product, some energy must be put in to reach the transition state.
This is called the activation energy. The activation energy is used to
break or weaken bonds in the substrates. Figure 4 shows these energy
374
8.1 MeTabOlisM
changes for an exergonic (energy releasing) reaction that is and is not
catalysed by an enzyme.
(a) transition state (b)
activation transition state
energy activation
energy
substrate
energy substrate
energy
product product
progress of reaction progress of reaction
Figure 4 Graphs showing activation energy (a) without an enzyme and (b) with Figure 5 A molecular model o the restriction
an enzyme enzyme EcoRV (purple and pink) bound
to a DNA molecule (deoxyribonucleic acid,
When an enzyme catalyses a reaction, the substrate binds to the active yellow and orange) . Restriction enzymes,
site and is altered to reach the transition state. It is then converted into also known as restriction endonucleases,
the products, which separate from the active site. This binding lowers recognize specifc nucleotide sequences and
the overall energy level of the transition state. The activation energy of cut the DNA at these sites. They are ound in
the reaction is therefore reduced. The net amount of energy released by bacteria and archaea and are thought to have
the reaction is unchanged by the involvement of the enzyme. However evolved as a deence against viral inection
as the activation energy is reduced, the rate of the reaction is greatly
increased, typically by a factor of a million or more. TOK
Types of enzyme inhibitors To wht xtnt houd thc
contrn th dvopmnt of
Enzyme inhibitors can be competitive or non-competitive. knowdg n cnc?
Some chemical substances bind to enzymes and reduce the activity of Sarin was a chemical developed as
the enzyme. They are therefore known as inhibitors. The two main types an insectide beore being applied is a
are competitive and non-competitive inhibitors. chemical weapon. It is a competitive
inhibitor o the neurotransmitter
Competitive inhibitors interfere with the active site so that the substrate acetylcholinesterase. Chemical
cannot bind. Non-competitive inhibitors bind at a location other than weapons would not exist without the
the active site. This results in a change of shape in the enzyme so that activities o scientists. In act, the name
the enzyme cannot bind to the substrate. Table 1 shows examples of Sarin is an acronym o the surnames o
each type. the scientists who frst synthesized it.
substrate Fritz Haber received the 1918 Nobel
Prize or Chemistry or his work in
competitive non-competitive developing the chemistry behind the
inhibitor inhibitor industrial production o ammonia
ertilizer. Some scientists boycotted the
active site is blocked binding of inhibitor no inhibition award ceremony because Haber had
by competitor changes shape of been instrumental in encouraging and
active site developing the use o chlorine gas in
Figure 6 the First World War. Haber is quoted as
saying: "During peace time a scientist
belongs to the World, but during war
time he belongs to his country."
375
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
enzym sutrat inhtor bndng
para-aminobenzoate
dihydropteroate suladiazine The inhibitor binds
synthetase O- reversibly to the
CO N enzymes active
SO2 site. While it remains
H2N bound, substrates
NN cannot bind. This is
H competitive inhibition.
H2N
The inhibitor binds
phosphoructokinase ructose-6-phosphate xylitol-5-phosphate reversibly to a site
away rom the active
P OH OH OH site. While it remains
bound, the active
CH2 CH2 C HC P site is distorted and
O substrate cannot bind.
H2C H C H CH2 This is non-competitive
inhibition.
C C OH OH
H HO
H OH
C C
OH H
Table 1 Examples of each type of inhibitor
Efects o enzyme inhibitors
Distinguishing diferent types o inhibition rom graphs at specied
substrate concentration.
Figure 7 represents the effect of substrate inhibitor prevents some of the enzymes from
concentration on the rate of an enzyme controlled being able to react regardless of substrate
reaction. concentration. Those enzymes that do not
bind inhibitors follow the same pattern as the
The orange line represents the effect of substrate normal enzyme. It takes approximately the same
concentration on enzyme activity in the absence concentration of enzyme to reach the maximum
of an inhibitor. rate, but the maximum rate is lower than the
uninhibited enzyme.
The red line shows the effect of substrate
concentration on the rate of reaction when maximum rate normal enzyme
a competitive inhibitor is present. When the of reaction
concentration of substrate begins to exceed the
amount of inhibitor, the maximum rate of the rate of reaction competitive inhibitor
uninhibited enzyme can be achieved; however, it
takes a much higher concentration of substrate to non-competitive inhibitor
achieve this maximum rate.
substrate concentration
The blue line shows the effect of substrate
concentration on the rate of reaction when a Figure 7
non-competitive inhibitor is present. In the
presence of a non- competitive inhibitor, the
enzyme does not reach the same maximum
rate because the binding of the non-competitive
376
8.1 MeTabOlisM
End-product inhibition active site no longer initial substrate
binds to threonine (threonine)
Metabolic pathways can be controlled by end-product
inhibition. threonine
in active site
Many enzymes are regulated by chemical substances that bind to special
sites on the enzyme away rom the active site. These are called allosteric enzyme 1
interactions and the binding site is called an allosteric site. In many (threonine
cases, the enzyme that is regulated catalyses one o the rst reactions in deaminase)
a metabolic pathway and the substance that binds to the allosteric site
is the end product o the pathway. The end product acts as an inhibitor. intermediate A
The pathway works rapidly in cells with a shortage o end product but enzyme 2
can be switched o completely in cells where there is an excess.
intermediate B
To see why this is such an economical way to control metabolic enzyme 3
pathways, we need to understand how the concentration o the product
o a reaction can infuence the rate o reaction. Reactions oten do not intermediate C
go to completion instead an equilibrium position is reached with a enzyme 4
characteristic ratio o substrates and products. So, i the concentration o
products increases, a reaction will eventually slow down and stop. This intermediate D
eect reverberates back through a metabolic pathway when the end enzyme 5
product accumulates, with all the intermediates accumulating. End-
product inhibition prevents this build-up o intermediate products. end product
( isoleuci ne)
An example of end-product inhibition isoleucine feedback inhibition
in allosteric
End-product inhibition o the pathway that converts site
threonine to isoleucine.
Figure 8
Through a series o ve reactions, the amino acid threonine is
converted to isoleucine. As the concentration o isoleucine builds up,
it binds to the allosteric site o the rst enzyme in the chain, threonine
deaminase, thus acting as a non-competitive inhibitor (gure 8) .
Investigating metabolism through bioinformatics
Developments in scientifc research ollow improvements in computing:
developments in bioinormatics, such as the interrogation o databases, have
acilitated research into metabolic pathways.
Computers have increased the capacity o scientists signicantly alter metabolic activity. S cientists
to organize, store, retrieve and analyse biological looking to develop new drugs test massive
data. Bioinormatics is an approach whereby multiple libraries o chemicals individually on a range
research groups can add inormation to a database o related organisms. For each organism a
enabling other groups to query the database. range o target sites are identied and a range
o chemicals which are known to work on
One promising bioinormatics technique that those sites are tested. One researcher called
has acilitated research into metabolic pathways chemogenomics the chemical universe tested
is reerred to as chemogenomics. Sometimes against the target universe.
when a chemical binds to a target site, it can
377
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
Chemogenomics applied to malaria drugs
Use of databases to identify potential new anti-malarial drugs.
Malaria is a disease caused by the pathogen a chloroquine-sensitive 3D7 strain and the
Plasmodium falciparum. The increasing resistance chloroquine-resistant K1 strain to see i these
o P. falciparum to anti-malarial drugs such as chemicals inhibited metabolism. Other related and
chloroquine, the dependence o all new drug unrelated organisms, including human cell lines,
combinations on a narrow range o medicines and were also screened. One promising outcome was
increasing global eorts to eradicate malaria all the identifcation o 1 9 new chemicals that inhibit
drive the need to develop new anti-malarial drugs. the enzymes normally targeted by anti-malarial
drugs and 1 5 chemicals that bind to a total o 61
Plasmodium falciparum strain 3 D 7 is a variety o dierent malarial proteins. This provides other
the malarial parasite or which the genome has scientists with possible lines o investigation in the
been sequenced. In one study, approximately search or new anti-malarials.
31 0,000 chemicals were screened against
Calculating rates of reaction
Calculating and plotting rates of reaction from raw experimental results.
A large number o dierent protocols are available substrate or the rate o appearance o a product.
or investigating enzyme activity. D etermining the Sometimes this will require conversion o units to
rate o an enzyme-controlled reaction involves yield a rate unit which should include s-1.
measuring either the rate o disappearance o a
Data-basd qustions: The efectiveness o enzymes
The degree to which enzymes increase the rate 2 State which enzyme catalyses its reaction at
o reactions varies greatly. B y calculating the
ratio between the rate o reactions with and the most rapid rate. [1 ]
without an enzyme catalyst, the afnity between
an enzyme and its substrate can be estimated. 3 Calculate the ratios between the rate o
Table 2 shows the rates o our reactions with
and without an enzyme. The ratio between these reaction with and without an enzyme or
rates has been calculated or one o the reactions.
ketosteroid isomerase, nuclease and
OMP decarboxylase. [3]
4 Discuss which o the enzymes is the more
1 State which enzyme catalyses the reaction eective catalyst. [3]
with the slowest rate in the absence o an 5 Explain how the enzymes increase the
rate o the reactions that they catalyse.
enzyme. [1 ] [2]
enzym Rat without Rat with Ratio btwn rat with
n zy m /s 1 n zy m /s 1 and without nzym
Carbonic anhydrase
Ketosteroid isomerase 1.3 101 1.0 106 7.7 106
Nuclease 1.7 107 6.4 104
OMP decarboxylase 1.7 1013 9.5 106
Table 2 2.8 1016 3.9 108
378
8.1 MeTabOlisM
Dt-d quton: Calculating rates of reaction actvty
22.0 For each o the ollowing
enzyme experiments,
21.5 describe how the rate o
reaction can be determined:
o xy ge n /% 21.0
20.5 ) Paper discs soaked in
the enzyme catalase
20.0 are added to diferent
concentrations o
19.5 hydrogen peroxide.
The reaction produces
19.0 oxygen bubbles.
18.5
) Lipase catalyses
18.0 10 20 30 40 50 60 70 the breakdown o
0 time/s triglycerides to atty
acids and water. The pH
51C 4C 21C 34C o the reaction solution
will lower as the reaction
Figure 9 Percentage of oxygen concentration over time at various temperatures after proceeds.
adding catalase to a 1.5% hydrogen peroxide solution
c) Papain is a protease that
Ten drops o a commercial catalase solution were added to our can be extracted rom
reaction vessels containing a 1 .5% hydrogen peroxide solution. Each pineapple ruits. Gelatin
o the solutions had been kept at a dierent temperature. The % cubes will be digested
oxygen in the reaction vessel was determined using a data logger in a by papain.
set-up similar to fgure 1 0.
d) Catechol oxidase
Figure 10 converts catechol to a
1 Explain the variation in the % oxygen at time zero. yellow pigment in cut
2 Determine the rate o reaction at each temperature using the graph. ruit. It can be extracted
3 Construct a scatter plot o reaction rate versus temperature. rom bananas. The
yellow pigment reacts
with oxygen in the air to
turn brown.
379
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
8.2 Cell respiration
Understanding Applications
Cell respiration involves the oxidation and Electron tomography used to produce images
reduction o compounds. o active mitochondria.
Phosphorylation o molecules makes them Skills
less stable.
Analysis o diagrams o the pathways o aerobic
In glycolysis, glucose is converted to pyruvate. respiration to deduce where decarboxylation
and oxidation reactions occur.
Glycolysis gives a small net gain o ATP without
the use o oxygen. Annotation o a diagram to indicate the
adaptations o a mitochondrion to its unction.
In aerobic cell respiration pyruvate is
decarboxylated and oxidized. Nature of science
In the link reaction pyruvate is converted into Paradigm shits: the chemiosmotic theory led to
acetyl coenzyme A. a paradigm shit in the eld o bioenergetics.
In the Krebs cycle, the oxidation o acetyl
groups is coupled to the reduction o hydrogen
carriers, liberating carbon dioxide.
Energy released by oxidation reactions is
carried to the cristae o the inner mitochondrial
membrane by reduced NAD and FAD.
Transer o electrons between carriers in the
electron transport chain is coupled to proton
pumping.
In chemiosmosis protons difuse through ATP
synthase to generate ATP.
Oxygen is needed to bind with the ree protons
to orm water to maintain the hydrogen gradient.
The structure o the mitochondrion is adapted to
the unction it perorms.
Oxidation and reduction
Cell respiration involves the oxidation and reduction
o compounds.
Oxidation and reduction are chemical processes that always occur
together. This happens because they involve transfer of electrons from
one substance to another. O xidation is the loss of electrons from a
substance and reduction is the gain of electrons.
A useful example to help visualize this in the laboratory is in the
B enedicts test, a test for certain types of sugar. The test involves the
380
8.2 Cell RespiRaTiOn
use o copper sulphate solution, containing copper ions with a charge adenine base
o two positive (Cu2+) . Cu2+ oten imparts a blue or green colour to ribose sugar
solutions. These copper ions are reduced and become atoms o copper
by being given electrons. Copper atoms are insoluble and orm a red or phosphates
orange precipitate. The electrons come rom sugar molecules, which are
thereore oxidized. ribose sugar
nicotinamide base
Electron carriers are substances that can accept and give up electrons
as required. They oten link oxidations and reductions in cells. The Figure 1 Structure of NAD
main electron carrier in respiration is NAD (nicotinamide adenine
dinucleotide) . In photosynthesis a phosphorylated version o NAD
is used, NADP (nicotinamide adenine dinucleotide phosphate) . The
structure o the NAD molecule is shown in fgure 1 .
The equation below shows the basic reaction.
NAD + 2 electrons reduced NAD
The chemical details are a little more complicated. NAD initially has
one positive charge and exists as NAD+. It accepts two electrons in the
ollowing way: two hydrogen atoms are removed rom the substance
that is being reduced. One o the hydrogen atoms is split into a proton
and an electron. The NAD+ accepts the electron, and the proton (H+)
is released. The NAD accepts both the electron and proton o the other
hydrogen atom. The reaction can be shown in two ways:
NAD+ + 2 H+ + 2 electrons ( 2e) NADH + H+
NAD+ + 2H NADH + H+
This reaction demonstrates that reduction can be achieved by accepting
atoms o hydrogen, because they have an electron. Oxidation can
thereore be achieved by losing hydrogen atoms.
Oxidation and reduction can also occur through loss or gain o atoms
o oxygen. There are ewer examples o this in biochemical processes,
perhaps because in the early evolution o lie oxygen was absent rom
the atmosphere. A ew types o bacteria can oxidize hydrocarbons using
oxygen:
C 7H1 5C H3 + _1 O2 C 7H1 5C H2O H
2
n-octane n-octanol
Nitriying bacteria oxidize nitrite ions to nitrate.
N O - + _1 O2 NO -
2 2 3
Adding oxygen atoms to a molecule or ion is oxidation, because the
oxygen atoms have a high afnity or electrons and so tend to draw
them away rom other parts o the molecule or ion. In a similar way,
losing oxygen atoms is reduction.
Phosphorylation
Phosphorylation of molecules makes them less stable.
Phosphorylation is the addition o a phosphate molecule ( P O 3- ) to
4
an organic molecule. Biochemists indicate that certain amino acid
sequences tend to act as binding sites or the phosphate molecule on
proteins. For many reactions, the purpose o phosphorylation is to make
381
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
the phosphorylated molecule more unstable; i.e., more likely to react.
Phosphorylation can be said to activate the molecule.
The hydrolysis o ATP releases energy to the environment and is
thereore termed an exergonic reaction. Many chemical reactions in the
body are endergonic (energy absorbing) and thereore do not proceed
spontaneously unless coupled with an exergonic reaction that releases
more energy.
For example, depicted below is the frst reaction in the series o reactions
known as glycolysis.
Glucose Glucose-6-phosphate
ATP AD P
The conversion o glucose to glucose-6-phosphate is endergonic and the
hydrolysis o ATP is exergonic. B ecause the reactions are coupled, the
combined reaction proceeds spontaneously. Many metabolic reactions
are coupled to the hydrolysis o ATP.
Glycolysis and ATP
Glycolysis gives a small net gain of ATP without the
use of oxygen.
The most signifcant consequence o glycolysis is the production o
a small yield o ATP without the use o any oxygen, by converting
sugar into pyruvate. This cannot be done as a single-step process and
instead is an example o a metabolic pathway, composed o many small
steps. The frst o these may seem rather perverse: ATP is used up in
phosphorylating sugar.
Glucose Glucose-6-phosphate Fructose6phosphate Fructose-1 ,6-bisphosphate
ATP AD P ATP AD P
However, these phosphorylation reactions reduce the activation energy
required or the reactions that ollow and so make them much more
likely to occur.
Pyruvate is a product of glycolysis
In glycolysis, glucose is converted to pyruvate.
In the next step, the ructose bisphosphate is split to orm two
molecules o triose phosphate. Each o these triose phosphates is then
oxidized to glycerate-3-phosphate in a reaction that yields enough
energy to make ATP. This oxidation is carried out by removing
hydrogen. Note that it is hydrogen atoms that are removed. I only
hydrogen ions were removed (H+) , no electrons would be removed
and it would not be an oxidation. The hydrogen is accepted by NAD+,
which becomes NADH + H+. In the fnal stages o glycolysis, the
phosphate group is transerred to AD P to produce more ATP and also
pyruvate. These stages are summarized in the equation below, which
occurs twice per glucose.
382
8.2 Cell RespiRaTiOn
NAD+ NADH + H+
triose phosphate glyce rate - 3 - p ho sp hate
The fate of pyruvate reduced NAD Glucose ATP
Glycolysis
In aerobic cell respiration pyruvate is Pyruvate ATP
decarboxylated and oxidized.
S CoA
Two molecules o pyruvate are produced in glycolysis CO
per molecule o glucose. I oxygen is available, this Link reaction
pyruvate is absorbed into the mitochondrion, where it is reduced NAD
ully oxidized.
Acetyl CoA
reduced FAD
2CH3COCOOH + 5O2 6CO2 + 4H2O Electron transport
pyruvate
Oxidative reduced Krebs
As with glycolysis, this is not a single-step process. cycle
phosphorylation NAD
Chemiosmosis
Carbon and oxygen are removed in the orm o carbon
dioxide, in reactions called decarboxylations. The
oxidation o pyruvate is achieved by the removal o pairs ATP
o hydrogen atoms. The hydrogen carrier NAD+, and a
related compound called FAD, accept these hydrogen Figure 2 A summary of aerobic respiration
atoms and pass them on to the electron transport chain where oxidative
phosphorylation will occur. These reactions are summarized in fgure 2 .
O CoA-SH
CO
The link reaction CO
In the link reaction pyruvate is converted into CH3 CO2 NAD+ reduced NAD CH3
acetyl coenzyme A.
Figure 3 The link reaction
The frst step, represented by fgure 3, occurs ater the pyruvate, pyruvic acid
which has been produced in the cytoplasm, is shuttled into the N AD +
mitochondrial matrix. Once there, the pyruvate is decarboxylated
and oxidized to orm an acetyl group. Two high energy electrons are CO2 reduced NAD
removed rom pyruvate. These react with NAD+ to produce reduced acetyl-CoA
NAD. This is called the link reaction, because it links glycolysis with
the cycle o reactions that ollow. CoA citric acid (6C)
OAA (4C)
The Krebs cycle reduced NAD NAD+
reduced NAD
In the Krebs cycle, the oxidation of acetyl groups CO2
NAD+ NAD+
reduced NAD
is coupled to the reduction of hydrogen carriers.
This cycle has several names but is oten called the Krebs CO2
cycle, in honour o the biochemist who was awarded the
Nobel Prize or its discovery. The link reaction involves one FAD H 2
decarboxylation and one oxidation. There are two more
decarboxylations and our more oxidations in the Krebs cycle. FAD
I glucose as oxidized by burning in air, energy would ATP ADP+iP
be released as heat. Most o the energy released in the
oxidations o the link reaction and the Krebs cycle is Figure 4 Summary of the Krebs cycle
used to reduce hydrogen carriers (NAD+ and FAD) .
383
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
TOK The energy thereore remains in chemical orm and can be passed on
to the nal part o aerobic cell respiration: oxidative phosphorylation.
What kinds o explanations do
scientists ofer, and how do these For every turn o the cycle, the production o reduced NAD occurs three
explanations compare with those times, decarboxylation occurs twice and the reduction o FAD occurs
ofered in other areas o knowledge? once. O ne molecule o ATP is also generated.
Hans Krebs was awarded the Nobel Oxidative phosphorylation
Prize in 1953. The two nal paragraphs
o the lecture that he gave on this Energy released by oxidation reactions is carried to the
occasion are reproduced here. cristae o the mitochondria by reduced NAD and FAD.
The reactions othe cycle have been In aerobic respiration, there are several points where energy released by
ound to occur in representatives oxidation reactions is coupled to the reduction o mainly NAD but also
oall orms olie, rom unicellular FAD. Reduced NAD is produced during glycolysis, the link reaction and
bacteria and protozoa to the the Krebs cycle. FADH2 is produced during the Krebs cycle.
highest mammals. The study o The nal part o aerobic respiration is called oxidative phosphorylation,
intermediary metabolism shows because AD P is phosphorylated to produce ATP, using energy released
that the basic metabolic processes, by oxidation. The substances oxidized include the FADH2 generated in
in particular those providing energy the Krebs cycle and the reduced NAD generated in glycolysis, the link
and those leading to the synthesis reaction and the Krebs cycle. Thus these molecules are used to carry the
ocell constituents, are also shared energy released in these stages to the mitochondrial cristae.
by all orms olie.
The electron transport chain
The existence ocommon eatures
in dierent orms olie indicates Transer o electrons between carriers in the electron
some relationship between the transport chain is coupled to proton pumping.
dierent organisms, and according
to the concept oevolution The nal part o aerobic respiration is called oxidative phosphorylation,
these relations stem rom the because AD P is phosphorylated to produce ATP, using energy released by
circumstance that the higher oxidation. The main substance oxidized is reduced NAD.
organisms, in the course omillions
oyears, have gradually evolved The energy is not released in a single large step, but in a series o small
rom simpler ones. The concept steps, carried out by a chain o electron carriers. Reduced NAD and
oevolution postulates that living FADH2 donate their electrons to electron carriers. As the electrons are
organisms have common roots, and passed rom carrier to carrier, energy is utilized to transer protons across
in turn the existence ocommon the inner membrane rom the matrix into the intermembrane space.
eatures is powerul support or The protons then fow through ATP synthase down their concentration
the concept oevolution. The gradient providing the energy needed to make ATP.
presence othe same mechanism
oenergy production in all orms o Chemiosmosis
lie suggests two other inerences:
frstly that the mechanism o In chemiosmosis protons difuse through ATP synthase
energy production has arisen very to generate ATP.
early in the evolutionary process;
and secondly that lie, in its present The mechanism used to couple the release o energy by oxidation to ATP
orms, has arisen only once. production remained a mystery or many years, but is now known to be
chemiosmosis. This happens in the inner mitochondrion membrane. It
1 Outline the argument or is called chemiosmosis because a chemical substance (H+) moves across
similarities o metabolism as a membrane, down the concentration gradient. This releases the energy
evidence or evolution. needed or the enzyme ATP synthase to make ATP. The main steps in the
process are as ollows (also see gure 5) .
2 Are there any alternative
explanations or the similarities?
384
8.2 Cell RespiRaTiOn
NADH + H+ supplies pairs o hydrogen atoms to the rst carrier in the inner inter
chain, with the NAD+ returning to the matrix. mitochondrial membrane
matrix membrane space
The hydrogen atoms are split, to release two electrons, which pass NADH + H+
rom carrier to carrier in the chain. NAD+
Energy is released as the electrons pass rom carrier to carrier, and H+
three o these use this energy to transer protons (H+) across the inner 2e-
mitochondrial membrane, rom the matrix to the intermembrane space.
H+
As electrons continue to fow along the chain and more and more FADH 2 H+
protons are pumped across the inner mitochondrial membrane, a FAD
concentration gradient o protons builds up. This proton gradient is a
store o potential energy.
To allow electrons to continue to fow, they must be transerred H2O H+
H+
to a terminal electron acceptor at the end o the chain. In aerobic 2H+
O2-
respiration this is oxygen, which briefy becomes O , but then
2
combines with two H+ ions rom the matrix to become water. O2
Protons pass back rom the intermembrane space to the matrix H+
through ATP synthase. As they are moving down the concentration
gradient, energy is released and this is used by ATP synthase to ATP ADP
phosphorylate AD P. +Pi
The role of oxygen low H+ high H+
concentration concentration
Oxygen is needed to bind with the free protons to form
water to maintain the hydrogen gradient. Figure 5 Summary of oxidative
phosphorylation
Oxygen is the nal electron acceptor in the mitochondrial electron
transport chain. The reduction o the oxygen molecule involves both
accepting electrons and orming a covalent bond with hydrogen.
By using up hydrogen, the proton gradient across the inner
mitochondrial membrane is maintained so that chemiosmosis can
continue.
Dt-bd quto: Oxygen consumption by mitochondria
Figure 6 shows the results o an experiment in 1 Explain why oxygen consumption by the
which mitochondria were extracted rom liver
cells and were kept in a fuid medium, in which mitochondria could not begin unless
oxygen levels were monitored. Pyruvate was
added at point I on the graph, and ADP was pyruvate had been added. [3]
added at points II, III and IV.
2 Deduce what prevented oxygen [2]
I II consumption between points I and II.
100
oxygen saturation / % 3 Predict, with reasons, what would have [2]
III happened i ADP had not been added at
point III.
50
IV 4 Discuss the possible reasons or oxygen [3]
consumption not being resumed ater
0 AD P was added at point IV.
time
Figure 6 Results of oxygen consumption experiment
385
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
The chemiosmotic theory
Paradigm shits: the chemiosmotic theory produced a paradigm shit in the feld
o bioenergetics.
In 1 961 Peter Mitchell proposed the chemiosmotic to destruction, ipossible. Meanwhile, the creator oa
hypothesis to explain the coupling o electron theory may have a very lonely time, especially ihis
transport in the inner mitochondrial membrane to colleagues fnd his views onature unamiliar and
ATP synthesis. His hypothesis was a radical departure difcult to appreciate.
rom previous hypotheses and only ater many years
was it generally accepted. He was awarded the Nobel The fnal outcome cannot be known, either to the
Prize or Chemistry in 1 978 and part o the Banquet originator o a new theory, or to his colleagues
Speech that he gave is reproduced here: and critics, who are bent on alsiying it. Thus, the
scientifc innovator may eel all the more lonely
Emile Zola described a work oart as a corner onature and uncertain.
seen through a temperament. The philosopher Karl
Popper, the economist F.A. Hayek and the art historian On the other hand, aced with a new theory, the
K.H. Gombrich have shown that the creative process members o the scientifc establishment are oten
in science and art consists otwo main activities: an more vulnerable than the lonely innovator. For, i
imaginative jumping orward to a new abstraction or the innovator should happen to be right, the ensuing
simplifed representation, ollowed by a critical looking upheaval o the established order may be very painul
back to see how nature appears in the light othe new and uncongenial to those who have long committed
vision. The imaginative leap orward is a hazardous, themselves to develop and serve it. Such, I believe, has
unreasonable activity. Reason can be used only when been the case in the feld o knowledge with which my
looking critically back. Moreover, in the experimental work has been involved. Naturally I have been deeply
sciences, the scientifc raternity must test a new theory moved, and not a little astonished, by the accidents o
ortune that have brought me to this point.
Structure and function in the Examine fgure 7 showing an electron micrograph
mitochondrion o a mitochondrion and a drawing representing
that mitochondrion.
The structure o the mitochondrion is
adapted to the unction it perorms. The mitochondrion is a semi-autonomous organelle
in that it can grow and reproduce itsel but it
There is oten a clear relationship between the still depends on the rest o the cell or resources
structures o the parts o living organisms and the and is otherwise part o the cellular system. 70S
unctions they perorm. This can be explained ribosomes and a naked loop o DNA are ound
in terms o natural selection and evolution. The within the mitochondrial matrix.
mitochondrion can be used as an example. I
mitochondrial structure varied, those organisms The mitochondrion is the site o aerobic respiration.
with the mitochondria that produced ATP most The outer mitochondrial membrane separates the
efciently would have an advantage. They contents o the mitochondrion rom the rest o
would have an increased chance o survival and the cell creating a compartment specialized or the
would tend to produce more ospring. These biochemical reactions o aerobic respiration.
ospring would inherit the type o mitochondria
that produce ATP more efciently. I this trend The inner mitochondrial membrane is the site o
continued, the structure o mitochondria would oxidative phosphorylation. It contains electron
gradually evolve to become more and more transport chains and ATP synthase, which carry
efcient. This is called adaptation a change in out oxidative phosphorylation. Cristae are tubular
structure so that something carries out its unction projections o the inner membrane which increase the
more efciently. surace area available or oxidative phosphorylation.
The intermembrane space is the location where
protons build up as a consequence o the electron
386
8.2 Cell RespiRaTiOn
transport chain. The proton build-up is used to The matrix is the site o the Krebs cycle and the link
produce ATP via the ATP synthase. The volume o reaction. The matrix fuid contains the enzymes
the space is small, so a concentration gradient across necessary to support these reaction systems.
the inner membrane can be built up rapidly.
Annotating a diagram of a mitochondrion
Annotation o a diagram to indicate the adaptations o a mitochondrion to its unction.
Outer mitochondrial membrane
separates the contents of the mitochondrion Matrix
from the rest of the cell, creating a cellular contains enzymes for the
compartment with ideal conditions for Krebs cycle and the link reaction
aerobic respiration Intermembrane space
Proteins are pumped
Inner mitochondrial into this space by the
membrane contains electron transport chain.
electron transport The space is small so the
chanins and ATP synthase concentration builds up
Cristae are projections of the inner membrane Ribosome quickly
DNA
which increase the surface area available for for expression of
oxidative phosphorylation
mitochondrial genes
Figure 7
actvty
0.1m
a) b) c) d)
Figure 8 Electron micrographs of mitochondria: (a) from a bean plant (b) from mouse liver (c) from axolotl sperm (d) from bat pancreas
Study the electron micrographs in gure 8 and then 80S ribosomes. Which o these hypotheses is
answer the multiple-choice questions. consistent with this observation?
1 The fuid-lled centre o the mitochondrion is called (i) Protein is synthesized in the mitochondrion.
the matrix. What separates the matrix rom the
cytoplasm around the mitochondrion? (ii) Ribosomes in mitochondria have evolved rom
ribosomes in bacteria.
) One wall. c) Two membranes. (iii) Ribosomes are produced by aerobic cell respiration.
b) One membrane. d) One wall and one membrane. ) (i) only c) (i) and (ii)
2 The mitochondrion matrix contains 70S ribosomes, b) (ii) only d) (i), (ii) and (iii)
whereas the cytoplasm o eukaryotic cells contains
387
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
Mitochondrial membranes are dynamic
Electron tomography used to produce images o active mitochondria.
Ideas in science sometimes change gradually. B ut are not simple inoldings but are invaginations,
sometimes they remain stable or years or even dening micro-compartments in the organelle.
decades and then undergo a sudden change. The cristae originate at narrow openings (crista
This can be due to the insight or enthusiasm o a junctions) that likely restrict diusion o proteins
particular scientist, or team. and metabolites between the compartments.
The membranes are not only very fexible but
The development o new techniques can also dynamic, undergoing usion and ssion
sometimes be the stimulus. The technique in response to changes in metabolism and
o electron tomography has recently allowed physiological stimuli.
three-dimensional images o the interior o
mitochondria to be made. One o the leaders in The working hypothesis is that the observed changes
this feld is D r. C armen Mannella, ormer D irector, in membrane shape (topology) are not random and
D ivision o Molecular Medicine, Wadsworth passive but rather a specic mechanism by which
C enter, Albany NY: Resource or Visualization o mitochondrial unction is regulated by changes in
B iological C omplexity. He recently gave this brie internal diusion pathways, e.g., allowing more
comment on developments in our understanding ecient utilization oADP. It appears that there
o mitochondrial structure and unction. are specic proteins and lipids that actively regulate
the topology othe inner membrane. This is a bit
The new take-home message about the speculative at the time but it gives a sense owhere
mitochondrial inner membrane is that the cristae things are headed in the eld.
Figure 9 Three images of the inner mitochondrial membrane of mitochondria from liver cells
show the dynamic nature of this membrane
TOK activity
There are some scientic Answer the ollowing questions with respect to the three images in gure 9 .
elds that depend entirely
upon technology or their ) The diameter o the mitochondrion was 700 nm. Calculate the [3]
existence, or example, magnication o the image.
spectroscopy, radio or X-ray
astronomy. What are the b) Electron tomography has shown that cristae are dynamic structures
knowledge implications o
this? Could there be problems and that the volume o the intracristal compartment increases when the
o knowledge that are
unknown now, because the mitochondrion is active in electron transport. Suggest how electron transport
technology needed to reveal
them does not exist yet? could cause an increase in the volume o fuid inside the cristae. [2]
c) Junctions between the cristae and boundary region o the inner [2]
mitochondrial membrane can have the shape o slots or tubes and
can be narrow or wide. Suggest how narrow tubular connections could
help in ATP synthesis by one o the cristae in a mitochondrion.
388
8.3 phOTOsynThesis
8.3 potot
Understanding Applications
Light-dependent reactions take place in the Calvins experiment to elucidate the
intermembrane space o the thylakoids. carboxylation o RuBP.
Reduced NADP and ATP are produced in the Skills
light-dependent reactions.
Annotation o a diagram to indicate the
Light-independent reactions take place in the adaptations o a chloroplast to its unction.
stroma.
Nature of science
Absorption o light by photosystems generates
excited electrons. Developments in scientifc research ollow
improvements in apparatus: sources o 14C and
Photolysis o water generates electrons or use autoradiography enabled Calvin to elucidate
in the light-dependent reactions. the pathways o carbon fxation.
Transer o excited electrons occurs between
carriers in thylakoid membranes.
Excited electrons rom Photosystem II are used
to generate a proton gradient.
ATP synthase in thylakoids generates ATP using
the proton gradient.
Excited electrons rom Photosystem I are used
to reduce NADP.
In the light-independent reactions a carboxylase
catalyses the carboxylation o ribulose
bisphosphate.
Glycerate 3-phosphate is reduced to triose
phosphate using reduced NADP and ATP.
Triose phosphate is used to regenerate RuBP
and produce carbohydrates.
Ribulose bisphosphate is reormed using ATP.
The structure o the chloroplast is adapted to its
unction in photosynthesis.
Location of light-dependent reactions
Light-dependent reactions take place in the
intermembrane space o the thylakoids.
Research into photosynthesis has shown that it consists of two very
different parts, one of which uses light directly (light-dependent
reactions) and the other does not use light directly (light-independent
389
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
reactions) . The light-independent reactions can only carry on in
darkness or a ew seconds because they depend on substances produced
by the light-dependent reactions which rapidly run out.
The chloroplast has an outer membrane and an inner membrane. The
inner membrane encloses a third system o interconnected membranes
called the thylakoid membranes. Within the thylakoid is a compartment
called the thylakoid space.
The light-dependent reactions take place in the thylakoid space and
across the thylakoid membranes.
Data-based questions: Freeze-fracture images of chloroplasts
I chloroplasts are rozen rapidly in liquid 4 Other membranes visible in the electron
nitrogen and then split, they racture across micrograph contain a variety o other
planes o weakness. These planes o weakness structures. Use the inormation on the
are usually the centres o membranes, between ollowing pages to deduce what these are. [3]
the two layers o phospholipid, where there are
no hydrogen bonds attracting water molecules to
each other. Structures within the membrane such
as the photosystems are then visible in electron
micrographs (see fgure 1 ) .
1 Describe the evidence, visible in the [2]
electron micrograph, or chloroplasts
having many layers o membrane.
2 Explain how photosystems become [2]
visible as lumps in reeze-racture
electron micrographs o chloroplasts.
3 Some membranes contain large particles
arranged in rectangular arrays. These are
Photosystem II. They have a diameter o
1 8 nm. Calculate the magnifcation o the Figure 1 Freeze-fracture electron micrograph
of spinach chloroplast
electron micrograph. [3]
The products of the light-dependent reactions
Reduced NADP and ATP are produced in the
light-dependent reactions.
Light energy is converted into chemical energy in the orm o ATP and
reduced NAD P in the light reacations. The ATP and reduced NAD P serve
as energy sources or the light-independent reactions.
The location of the light-independent reactions
Light-independent reactions take place in the stroma.
The inner membrane o the chloroplast encloses a compartment called
the stroma. This is a thick protein-rich medium containing enzymes
or use in the light-independent reactions, also known as the Calvin
390
8.3 phOTOsynThesis
cycle. In the light-independent reactions the Calvin cycle is an anabolic
pathway that requires endergonic reactions to be coupled to the
hydrolysis o ATP and the oxidation o reduced NAD P.
Figure 2 summarizes the processes o both the light-dependent and light-
independent reactions.
outer membrane of chloroplast
inner membrane
of chloroplast
thylakoid membrane CO2
thylakoid space
light P1 + ADP
energy H2O
ATP Calvin
Figure 2 light-independent reactions NADP cycle
- photolysis NADPH + H+
- photoactivation su ga rs
- electron transport 2e-
- chemiosmosis
- ATP synthesis 2H+ + 1 O2
- reduction of NADP 2
light-independent reactions
- carbon xation
- carboxylation of RuBP
- production of triose phosphate
- ATP and NADPH as energy sources
- ATP used to regenerate RuBP
- ATP used to produce carbohydrates
Photoactivation
Absorption of light by photosystems generates
excited electrons.
Chlorophyll and the accessory pigments are grouped together in large
light-harvesting arrays called photosystems. These photosystems are
located in the thylakoids, an arrangement o membranes inside the
chloroplast. There are two types o light-harvesting arrays, called
Photosystems I and II. In addition to light-harvesting arrays, the
photosystems have reaction centres (fgure 3) .
Both types o photosystem contain many chlorophyll molecules, which
absorb light energy and pass it to two special chlorophyll molecules in
the reaction centre o the photosystem. Like other chlorophylls, when
these special chlorophyll molecules absorb the energy rom a photon o
light an electron within the molecule becomes excited. The chlorophyll
is then photoactivated. The chlorophylls at the reaction centre have
the special property o being able to donate excited electrons to an
electron acceptor.
391
8 M ETABOLI SM , CELL RESPIRATION AN D PHOTOSYN TH ESI S ( AH L)
Photosystem II Rather conusingly, Photosystem II, rather than Photosystem
light light harvesting array reaction centre I, is where the light-dependent reactions o photosynthesis
begin. The electron acceptor or this photosystem is called
plastoquinone. It collects two excited electrons rom
Photosystem II and then moves away to another position in
the membrane. Plastoquinone is hydrophobic, so although it
is not in a xed position, it remains within the membrane.
primary plastoquinone Absorption o two photons o light causes the production
acceptor o one reduced plastoquinone, with one o the chlorophylls
transfer of at the reaction centre having lost two electrons to a
e- electrons plastoquinone molecule. Photosystem II can repeat this
pigment process, to produce a second reduced plastoquinone, so the
molecules chlorophyll at the reaction centre has lost our electrons and
two plastoquinone molecules have been reduced.
chlorophyll molecules Photolysis
that transfer electrons
Photolysis of water generates electrons for use in the
Figure 3 Diagram showing the relationship light-dependent reactions.
between the light-harvesting array, the
reaction centre and plastoquinone Once the plastoquinone becomes reduced, the chlorophyll in the
reaction centre is then a powerul oxidizing agent and causes the water
molecules nearest to it to split and give up electrons, to replace those
that it has lost:
2H2O O2 + 4H+ + 4e-
The splitting o water, called photolysis, is how oxygen is generated
in photosynthesis. O xygen is a waste product and diuses away.
The useul product o Photosystem II is the reduced plastoquinone,
which not only carries a pair o electrons, but also much o the energy
absorbed rom light. This energy drives all the subsequent reactions o
photosynthesis.
Figure 4 Electron micrograph of The electron transport chain
thylakoids 75,000
Transfer of excited electrons occurs between carriers in
thylakoid membranes.
The production o ATP, using energy derived rom light is called
photophosphorylation. It is carried out by the thylakoids. These are
regular stacks o membranes, with very small fuid-lled spaces inside
(see gure 4) . The thylakoid membranes contain the ollowing structures:
Photosystem II
ATP synthase
a chain o electron carriers
Photosystem I.
Reduced plastoquinone is needed, carrying the pair o excited electrons
rom the reaction centre o Photosystem II. Plastoquinone carries the
electrons to the start o the chain o electron carriers.
392
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