1

MODEL ANSWERS FOR BS934 JAN 2008 EXAM

SECTION A
QUESTION 1
Learning objectives covered

Understand which variables are important in the optimisation of a PCR.

List and describe other PCR-based methods, and discuss how these can be applied.
Describe how nucleic acids can be visualised and to appreciate how different gel types can be
used to discriminate between molecules of different sizes.
List and describe other PCR-based methods, and discuss how these can be applied.

Model Answer
1. By using real time PCR, you need to compare the relative levels of messenger RNA (mRNA)
encoding the Bax1 inhibitor protein in an immortalised human cell line compared with the primary
tissue from which it was derived.
You are provided with cDNA samples that have been prepared from total mRNA from the primary
tissue and cell line
At the end of the reaction, the real time PCR machine has given you the following fluorescent data for
cycles 15 25 for your primer pair and also that of a standard gene (Actin) with which to normalise
data between samples. The data are as follows:
Cycle
Actin
Actin
Bax1
Bax1
Number (cell line) (tissue) (tissue) (cell line)
15
16
17
18
19
20
21
22
23
24
25

0
0.01
0.01
0.05
0.09
0.17
0.29
0.49
0.81
1.25
1.80

0
0
0
0.01
0.02
0.04
0.07
0.13
0.27
0.53
0.94

0
0
0.01
0.01
0.02
0.04
0.09
0.17
0.33
0.66
1.30

0.04
0.09
0.19
0.38
0.62
1.00
1.48
1.96
2.36
2.67
2.89

(c) Using the graph paper provided, determine the threshold cycle (Ct) value, to the nearest
whole number, and the cycle efficiency (E), to two decimal places, of each reaction.

fluorescent values (arbritary units

[40%]
3.1
3
2.9
2.8
2.7
2.6
2.5
2.4
2.3
2.2
2.1
2
1.9
1.8
1.7
1.6
1.5
1.4
1.3
1.2
1.1
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1
-0.2

Actin (tissue)
Actin (cell line)
Bax1 (tissue)
Bax1 (cell line)
Threshold
fluorescence for
calling Ct

10

20

30

cycle number
Answer (i) The Ct values ought to be as follows (although I will allow some leeway in choice of
threshold fluorescence value) Graph (10 marks), deduct marks for incorrect axis labelling and failure
to show where threshold fluorescence value is chosen
Bax1 (cell line) = 17; Bax1 (tissue) = 22; Actin (cell line) = 21; Actin (tissue) = 23.(2. 5 marks for
each correct answer)
Deduct 0.5 mark for not rounding any figures to whole numbers.
Cycle
Actin
Actin
Bax1
Bax1
Number (cell line) (tissue) (tissue) (cell line)
15
16
17
18
19
20
21
22
23

-2
-2
-1.3
-1.05
-0.77
-0.54
-0.31
-0.09

-2
-1.7
-1.4
-1.15
-0.89
-0.57

-2
-2
-1.7
-1.4
-1.05
-0.77
-0.48

-1.4
-1.05
-0.72
-0.42
-0.21
0
0.17
0.29
0.37

3
0.1
0.26

24
25

-0.28
-0.02

0.43
0.46

-0.18
0.11

To answer second part of Q3 on efficiency they should convert fluorescence data to log10 values so they
can see which part of the graph is linear. They can then plot this as a semi-log plot and determine from
slope or just simply directly from the calculated values on the table. The log fluorescence
increase/cycle = logE. Convert back to natural numbers.
The numbers underlined can be used to calculate a log increase per cycle, which once they
know that from a plot or direct calculation from the log values is the most direct route: There is some
variation in the choice of straight line which will affect the numbers but the calculations should be
clear.( For graph or table produced and clear indication of which cycles values are taken for
calculation of E = up to 10 marks)
Bax1 (cell line) log E = -0.42 (-1.4)/4 = 0.98/4 = 0.245. Therefore, E = 1.76
Bax1 (tissue) log E = 0.11 (-1.7)/7 = 1.81/7 = 0.259. Therefore, E = 1.82
Actin (cell line) log E = -0.02 (-1.7)/7 =1.68/7 =0.24. Therefore, E = 1.74
Actin (tissue) log E = -0.31 (-1.3)/5 = 0.99/5 = 0.198. Therefore, E = 1.58
(2.5 marks for each correct answer. Note there is some leeway in the numbers chosen.)

log 10 fluorescence valu

1
0.5
0
-0.5

10

20

-1

30

Actin (tissue)
Actin (cell line)
Bax1 (tissue)
Bax1 (cell line)

-1.5
-2
-2.5
cycle number

(d) What is the fold difference in expression of the Bax1 gene in the cell line compared with the
tissue from which it was derived, normalised for actin? Show your calculations.
[20%]
Ratios if cell line to tissue control bax1 over actin can be done in a number of ways:

XBax1/Xactin = ECt (bax tissue - bax cell line)/ ECt (act tissue - act cell line)
E values have to be averaged for both bax primers and actin primers
E (bax) = 1.76+ 1.82/2 = 1.79; E(act) = 1.74 + 1.58 /2 = 1.66
Note that it is the difference in the cycles irrespective of which is hi

XBax1/Xactin = 1.79(21-16)/1.66(21-19) = 1.795/1.662 = 18.4/2.8 = 6.6 fold.

Up to 9 marks each for correct calculation of actin and Bax1 (note that the calculations for both genes
can be done separately and then put as ratio at the endas long as calculations are shown this will be
acceptable) Total 18 marks. 2 marks for the expression of the answer as fold increase.
QUESTION 2
Learning objectives covered

Explain the principles of enzyme kinetics, including enzyme inhibition and define the terms
Michaelis constant, enzyme activity, specific activity, molar activity & catalytic efficiency, &
calculate the value of each from experimental data

(a) To answer this question it is necessary to know the Lineweaver-Burk equation or to be able to
derive it from the Michaelis-Menten.
I/V = Km/Vmax.1/[S] + 1/Vmax
The first step is to deduce reciprocal values at the different inhibitor concentrations for both substrate
concentration and the velocity data.
1/V (M-1 min)
1/[S] (M-1)
0.05
0.1
0.15
0.2

I=0

I=3

I=10

0.1

0.3

0.5

0.08

0.24

0.4

0.06

0.18

0.3

0.04

0.12

0.2

These data are used for the plot. The plot should be 1/V on the y axis versus 1/[S] on the x-axis as
follows:

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Lineweaver-Burk plot
y = 0.4x + 0.02

0.6
0.5

1/V (uM min)

0.4
Series1

-1

0.3

Series2

0.2

Series3
Linear (Series3)

0.1

Linear (Series2)
Linear (Series1)

0
-0.2

-0.1

0.1

0.2

0.3

-0.1
-0.2
-1

1/[S](uM )

The gradient (Km/Vmax), the intercept on the x-axis (-1/Km) and the intercept(s) on the y-axis
(1/Vmax and 1/Vmax) should be labelled. There should also be a title and the two axes should be
properly labelled with correct units. From the graph the Km ( 20 M) and the Vmax (50 M min-1) can
be determined
(b) The inhibitor does not affect the Km (x-intercept) but does change the velocity (y intercept). Thus
the inhibitor is non-competitive.
(c) The inhibitor is not binding at the active site as can be seen with the constant Km observed with the
different concentrations of inhibitor. However the Vmax of the enzyme is clearly decreasing with
increased amounts of inhibitor so the inhibitor must be binding elsewhere and thus altering the
enzymatic activity of the enzyme.
SECTION B
For each of the section B questions, Some leeway (10 marks) can also be given for good structure and
answers that are factually correct but miss the direct point of the question.
1. Describe the chemical structure of DNA and how this is exploited in the Southern blotting
procedure for analysing restriction enzyme digested genomic DNA from eukaryotic and
prokaryotic sources. In your answer you should include considerations of using a PCRgenerated probe which is a 1 kb double stranded DNA fragment which is 90%
homologous to the target sequence.
[100%]
Learning objectives covered

Distinguish between Southern and Northern hybridisation, describing their key features,
similarities and differences.
Distinguish between restriction fragment/PCR derived probes and oligonucleotide probes, and
discuss their application.

List and describe the key enzymes (and their actions) that are used today in molecular biology.
Outline the key stages in isotopic detection, and list the main radioisotopes (and molecules)
used in detection.
Describe how nucleic acids can be visualised and to appreciate how different gel types can be
used to discriminate between molecules of different sizes.
Distinguish between restriction fragment/PCR derived probes and oligonucleotide probes, and
discuss their application.

Answer notes
A good answer should contain the following ideas or concepts.
The only key features of DNA really needed that are relevant are that it in the context of the question
It will be double stranded (double helix) in which each strand is bound to the other primarily
by H bonding with complemenary bases (A-T and G-C). (5)
That G-C base pairs are more tightly bonded (3 H bonding links) compared with A-T (2 links)
Therefore GC rich DNA will have a higher melting temperature for denaturation than AT rich
DNA. (10)
This property will affect the ability to denature the DNA for blotting, the ability of the probe to
be denatured to single-stranded DNA, hybridise to its target by complementarity (up to 15, up
to 5 for each point)
The stringency of the washing (salt concentration, temp) will also dictate how well probe
hybridises (disruption of H-bonding)..Since probe is not 100% homologous this will affect
when the probe signal is washed off . They may mention 2xSSC versus 0.1 x SSC this is
acceptable for low versus high stringency respectively. (15)
Polymer assembled from constituent bases as nucleotides (important for one method of making
probe) (5)
The ends of the probe DNA will be 5hydroxylated as opposed to a normal DNA fragment
which will be 5 phosphorylated (10..because this would be well spotted and draws in
knowledge across the lecture series)
The other feature of DNA is the sequence of base pairs..this is perhaps more obscure (but
justified)they are using restriction enzymes which recognise specific often palindromic
sequenceseg EcoR1 (5GAATTC3) which will cut the DNA into a range of fragments that
can be separated by electrophoresis. (5)
Flow diagram of the procedure and/or a direct description of the procedure should be there
(from intranet material)thus digestion of the DNA .separation of fragments by
electrophoresispreparing the gel by alkaline denaturation which breaks double stranded
DNA backbone to single stranded, blot and immobilise to membrane by vacuum baking or UV
cross linking (electrostatic or covalent interactions mentioned then this extra marks and will be
evidence of extra reading). (up to 20 points, depending on detail and accuracy)
Probe preparation briefly described .common method random priming using mixed
hexamers which on heat denatured template are used to prime copying by DNA polymerase I
(Klenow fragment) and incorporate fluorescent or radioisotope labeled nucleotides into copied
fragments. Alternatively, the end of the fragment could be labeled with -32P-ATP using T4
polynucleotide kinase since the ends of the fragment are 5hydroxylated (up to 15 points
depending on detail and accuracy).

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2.

a) Describe how fluorescent cycle sequencing works and how you would use this method
as part of a sequencing strategy for an unknown 20kb cloned DNA fragment.
[40%]
Briefly indicate the advantages and disadvantages of your chosen sequencing strategy.
[10%]
b) For genome sequencing projects, explain using examples, how sequencing strategies
have evolved to cope with the large size of some genomes.
[50%]

Learning objectives covered

Appreciate how different vectors can be used to clone DNA of different sizes, and for different
applications.
Appreciate the diverse array of gene cloning strategies.
Be able to construct a restriction map of a plasmid from restriction fragment data.
Give an example of a technique used to map large fragments, and describe its key features
Appreciate how fluorescence based detection and automation have revolutionised DNA
sequencing, and enabled genome sequencing to become routine.
Describe the key features of Sanger sequencing
Discuss the advantages of fluorescence automated sequencing, and explain the key stages in the
sequencing and sequence assembly of a named microbial genome.
Describe the key features of a DNA array (here in the brief context of development of new
sequencing strategies).

Model Answer
a) Describe how fluorescent cycle sequencing works and how you would use this method as part of
a sequencing strategy for an unknown 20kb cloned DNA fragment.
[40%]
Briefly indicate the advantages and disadvantages of your chosen sequencing strategy.
[10%]
A good answer will contain the following:
There are a number of ways of tackling the question. The direct route would be to go straight to a
description of fluorescent cycle sequencing in which the four fluorescent dideoxy NTPS are in one
tube, there is Taq polymerase and it is essentially a linear PCR reaction using a single primer.
Alternatively, the candidate could describe conventional Sanger dideoxy sequencing and then briefly
show how fluorescent cycle sequencing differs. Either way there should be a description of the
following elements not necessarily expressed this way:
Up to 30 marks available for this
The use of a single-stranded DNA template either from M13 phage and/or denatured double
stranded DNA (as part of the PCR cycles used)
Depends on a sequence specific primer on which the DNA (Taq) polymerase begins to copy off
the template. The primer is an oligonucleotide specific to plasmid vector sequence near the
DNA fragment insertion site or specific to some part of the insert.
The use of chain terminating didexoynucleotides which prevent the insertion of a subsequent
nucleotide by the DNA polymerase.

The right mix of dNTPs and ddNTPs that statistically allow chain termination at every base in
some of the DNA molecules, generating a population of fluorescently labeled ssDNA fragments
that differ by one base in size.
Separated by denaturing acrylamide gel or capillaries on an automated sequencing machine,
such as the Applied Biosystems, Li-Cor, Amersham machines. As labeled fragments pass to the
bottom of the gel or capillary the due are excited and emission of the right wavelength detected.
A good description here would have included some reading about machinery and a precise
descpription of detectors and use of a laser
Each type of base (A, C, G, T) each has its own signature fluorescent dye (eg HEX, ROX, JOE,
TAMRA, NED, TET, 6-FAM, 5-FAM, R6G, R110) machines can excite with laser emitted
light with appropriate filters and emission wavelengths captured by fliters palced in front of
detectors. (5)

Sequencing strategies should describe one of the following: NOTE WELL answer could combine
these methods to get rid of disadvantagesthis is definitely allowed and would attract a good mark (
up to 10 marks for this)
1. Produce a restriction map, subclone fragments and sequence them from vector anchored
primers, generating fragments small enough to sequence both strands
2. Walk along the sequence in both strands starting from vector anchored primers.obtain
sequence data, design new primers, more sequence, new round of primers new sequence and so
on.
3. Shotgun strategy. Prepare fragment insert DNA from vector, sheer the DNA by sonication and
select fragments of a specific size (usually <1kb), insert into plasmid, pick colonies at random
and sequence.
Pros and cons (up to 10 marks)
1. Advantage.. links sequence to recognisable restriction sites, allowing sequence to be easily
assembled correctly. Disadvantage Slow, might not get fragments of right size for complete
coverage.
2. Advantage. Sure. Will get you there. Disadvantage Slow
3. Advantage. Rapid. Very good multiple coverage of sequence Disadvantage Can be subject to
law of diminishing returns, incomplete coverage, may require other method to fill gaps.
b) For genome sequencing projects, explain using examples, how sequencing strategies
have evolved to cope with the large size of some genomes.
[50%]
This can be done a number of ways, either written generically or going through a list of organisms
of increasing genome size.
A good answer will impose some order on the way this is done and have the following elements or
ideas conveyed: The best marks will be those who show clear evidence of having tapped in on
the extensive literature provided by providing a little more detail for each example
1. Improvements in sequencing technology (eg. transition to Sanger sequencing and then later the
transition to cycle sequencing with fluorescent dyes and semi-automation) (up to 5)

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2. That genome sequencing is now done by national facilities (eg TIGR, Sanger, Genethon) and
for really big projects (yeast, Arabidopsis, Drosophila, C.elegans and human) international
effort combining these national facilities (up to 5)
3. Sequencing strategies. (up to 30 marks can be awarded)
.first example was phage lambda (48.5kb; 1982), which was done by subcloning
restriction fragments and sequencing by both conventional 4 reaction didexoy (2.5)
then with cycle sequencing and automation with high throughput shotgun approaches
(random cloning shearing of DNA) allowed the first bacterial genome Haemophilus
influenzae 1.83Mbp. 1996 (up to 5)
they may mention intervening progress on organellar genomes, in which case give extra
marks (2.5)
Subsequently 225 bacterial genomes now sequenced, 21 archaea. Becoming almost
routine. (2.5)
Chromosomes assigned in international consortia for sequencing (up to 2.5)..Can be
separated by flow cytometry and individual BAC or YAC libraries made or BAC clones
can be assigned to chromosomes using FISH (5)
Bigger genomes such as Arabidopsis, human sequencing projects use a heirarchial
shotgun cloning strategy, in which a tiling path or physical link (contig) of overlapping
BAC/YAC clones are ordered across each chromosome to produce a physical map. This
is done by restriction mapping (using rare cutting enzymes), Southern hybridisation and
(most likely now) BAC end sequencing to look for overlaps. (Up to 5)
Yeast genome first international collaboration in which allocated chromosomes were
sequenced by specific consortia. Eg. EU labs did chromosome 4 of Arabidopsis.(up to
2.5)
Year 2000 first draft human genome sequence 3000Mbp and Arabidopsis 115Mbp (up
to 2.5)
Additional information
Latest developments include sequencing total DNA from the environment..eg Sargasso
Sea (Venter paper in Science should be discussed)..this is allowed by the declining costs
of sequencing as facilities mature, as automation improvesbut maybe of limited
success (see comments on Venter paper) (up to 5)
Finally they may mention and discuss pyrosequencing in which massively parallel
sequencing is done on short cloned fragments.
Technology works and is increasingly driving down sequencing costs for genome
sequencing. The maximum read is about 200 bases but is made up by very high throughput
and radom cloning and sequencing methods with no cloning steps required.
Section C
Question 5
Question 2: Discuss the principles and practice of column chromatography as applied to protein
purification [60%]. Discuss the IMAC method for affinity chromatography and its use for
purification of tagged proteins [40%].
Objectives tested

10
- Explain the underlying principles of commonly used chromatographic techniques for protein
purification;
- Describe how detergents and mechanical methods may be employed to extract soluble and
membrane proteins from eukaryotic and bacterial cells;
Answer
(a)
Column chromatography is a separation method based on the distribution of analytes between
two phases. A stationary phase is packed in a column of appropriate size. The proteins to be
separated are dissolved in certain volume of mobile phase and injected onto the column. As the
mobile phase flows through the column the proteins are separated based on their ability to
interact with the stationary phase. [5]
Several main types of chromatographic methods can be distinguished by the type of interaction
between the stationary phase and the analyte molecules:
o Ion-exchange chromatography exploits electrostatic interactions between the alternatively
charged stationary phase and analyte molecules. In anion exchange chromatography the
stationary phase is derivatized with groups bearing positive charges, which interact with the
negatively charged analyte molecules. Cation exchange chromatography uses negatively
charged stationary phase and is used for separation of positively charged compounds. Usually
gradients of high ionic strength solvents are used to elute the proteins sequentially from the
column. For example proteins with high negative charge would elute late from a cation
exchange column because a high concentration of negative ions is needed to screen the
electrostatic interaction of the protein with the stationary phase. Because of the specifics of ionexchange chromatography the initial sample has to contain very low concentration of
electrolytes [15].
o Size-exclusion chromatography, also called gel-filtration, separates compounds based on size
differences. Stationary phases consist of porous beads. The interaction between the analyte
(protein) molecules and the stationary phase effectively partitions the analyte molecules
according to their size as larger molecules are either completely excluded from the pores of the
stationary phase or can only enter to a very limited extend and elute from the column early.
Smaller molecules are retarded as they can go deeper into the pores of the stationary phase. Gel
filtration does not require buffer exchange and can be applied directly following ion-exchange
separations [10].
o Hydrophobic interaction chromatography is a technique that separates molecules on the base of
their hydrophobicity. In proteins the side chains of W, L, I, F , when exposed would interact
with other non-polar groups. This is called hydrophobic interaction. The force that drives this
interaction comes from the hydrophobic effect. The stationary phase for hydrophobic
interaction chromatography has non-polar groups attached and can separate proteins according
to the number of exposed non-polar amino acid side chains. Hydrophobic interaction is
enhanced by high concentration of salts. That is why this method is good follow-up to ionexchange chromatography [15].
o Affinity chromatography is based on molecular recognition. A ligand with high affinity to the
molecule of interest is attached to the stationary phase and allowed to interact with its target.
All other components of the sample flow through the column unrestricted and are washed away.
The bound molecules are eluted under conditions that decrease the affinity of the ligand for its
targets. Such conditions might be lower or higher pH, different ionic strength for example

11
many affinity interactions require some ions to be present and if the ionic strength of the mobile
phase is decreased they will be disrupted. In such cases bound molecules can be eluted by
simply flushing distilled water through the column. Examples of affinity chromatography are
antibody affinity methods where antibody are attached to the column to purify specific antigens
and enzyme substrate or inhibitor columns where small molecules with affinity to certain
enzymes are attached to the column [15].
(b)

The IMAC (immobilized metal affinity chromatography) is based on ligand exchange

mechanisms involving coordination of transition metal ions. This process is also known as
chelating and the resulting complexes are called metal chelates. Several O and N atoms of the
chelating ligand interact with the metal ion by donating one of their free electron pairs to its
unoccupied orbitals. [10]
o The most commonly used IMAC method for purification of tagged proteins is Ni affinity
chromatography. The stationary phase of the column contains the polydentate ligands NTA
(nitrilotetraacetic acid), or IDA (iminodiacetic acid), attached covalently to agarose or other
polymer. Polydentate means that one molecule of the ligand provides electrons to not 1 but
several orbitals in the coordination sphere of the metal ion. Only polydentate ligands can
chelate transition metal ions. This leads to a very tight binding of the metal ion. The Ni2+ ion
has 6 ligand binding sites (unoccupied orbitals) in its coordination sphere. When it is
complexed to NTA it uses 4 orbitals to bind the NTA matrix. The 2 remaining binding sites are
free and can interact with other ligands. Such ligands could be the side chains of histidine,
phosphorylated serine, threonine or tyrosine, the carboxyl groups of aspartate and glutamate
side chains, and the C terminus. [10]
o The general principle is chelating ligand is used to immobilize the metal ion on the column,
but an interaction with certain monodentate non chelating ligand should be the basis of the
affinity purification. In this way the metal ion stays on the column and is not displaced by the
ligand. IMAC can be used to purify tagged proteins if the tag contains appropriate ligand
groups. Usually such groups are the imidazole rings of the histidine side chains. At basic pH the
imidazole ring contains an N atom with a free electron pair. Thus a single Ni ion interacts with
2 histidine side chains. Proteins to be purified are tagged with peptides containing 6 or more
consecutive histidines so under basic pH they have a very high affinity for the Ni ion. Native
proteins can also interact but with lower affinity. To minimize this interaction a low
concentration of imidazole is maintained in the lysis buffers. Once the tagged protein is bound
the column is washed with buffer containing imidazole. Then the protein needs to be eluted.
[10]
o There are 2 methods for eluting his-tagged proteins from an IMAC column. A competitive
elution relies on displacing the bound protein by a high concentration of a ligand, usually
imidazole. The displacing agent should not be a very strong chelator, like EDTA for example,
since it would strip the metal ion from the column, which would contaminate the protein
preparation. The second method relies on the pH dependence of the ligand exchange
interaction. At low pH the imidazole ring becomes protonated and positively charged, which
makes impossible the interaction with the metal ion, also positively charged. An added
advantage of the IMAC method for purification of his-tagged proteins is that it can be used
under denaturing conditions, which can yield very highly purified proteins in a single step. [10]

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Question 6
Discuss the mechanism of the elongation stage of protein synthesis in prokaryotic organisms
[70%]. Explain how release factors regulate termination of translation in prokaryotic organisms
[30].
Objectives tested
- Discuss protein synthesis
Answer
(a)
Newly intact ribosome contains complete E, P & A sites, with P-site occupied by charged
formyl Met-tRNA. A-site is positioned over the second codon of mRNA. E site is unoccupied.
Elongation begins when a suitable charged tRNA molecule enters this site. [5]
o With both sites occupied, the two amino acids are in close contact, which allows for peptide
bond formation by the peptidyl trasferase activity of the ribosome. This is followed by action of
tRNA deacylase, which breaks the link between formylMet & its tRNA.
o Uncharged tRNAformylMet is then expelled from P-site to E site by a second (charged) tRNA, to
which the dipeptide is bound (i.e. translocation step).
o A third charged tRNA molecule enters the A-site & elongation cycle is repeated. [15]
o Elongation is controlled by three elongation factors: EF-Tu, EF-Ts, and EF-G. EF-Tu is a
GTPase (G-protein) and is the most abundant protein in E. coli. EF-Tu complexed to GTP binds
amino acyl-tRNA and caries it to the A site of the ribosome. At this step the codon of mRNA
and the anticodon of tRNA are paired, which triggers the hydrolysis of GTP bound to EF-Tu to
GDP. EF-Tu bound to GDP has much lower affinity to the ribosome and is therefore displaced
in the cytosol. EF-Tu has low intrinsic GTPase activity. It is stimulated only when the ternary
complex (EF-Tu bound to GTP and amino acyl-tRNA) interacts with the ribosome. [15]
o The EF-Tu bound to GDP then react with EF-Ts. EF-Ts s a nucleotide exchange factor. It
catalyzes the exchange of GDP in the active site of EF-Tu for GTP. This regenerates the EF-TuGTP complex and allows for another cycle of elongation. [5]
o The binding of correct tRNA at the A sites is ensured by specific molecular interactions
between rRNA, mRNA and the tRNA. The binding of mRNA and cognate tRNA in the A site
induces conformational changes in the rRNA, which enforce the interaction. This type
recognition is called induced fit. The conformational changes allows the ribosome to monitor
the pairing of the codon from the mRNA and the anticodon of the amino acylated tRNA a
proof reading mechanism. [15]
o The third elongation factor EF-G controls the translocation of the ribosome along the mRNA.
The structure of EF-G and EF-Tu are similar and they are both GTPases. However, EF-G does
not bind tRNAs. Instead it has a domain that resembles the tRNA. This is an example of
molecular mimicry and is important for the process of translocation. EF-G competes with EFTu for binding to the ribosome. Upon GTP hydrolysis EF-G undergoes a conformational
change, which allows it to insert its tRNA mimicking domain in the anticodon region of the
ribosomal A site. This forces the ribosome to slide relative mRNA and translocation occurs.
[15]
(b)

Termination begins when a stop codon enters the A site. Two proteins are involved in the
recognition of stop codons in bacteria. RF1 (Release factor 1) and RF2 both recognize UAA.

13
Only RF1 recognizes UAG and only RF2 recognizes UGA. Therefore UAA is much stronger
stop signal then the other two. [10]
o When a stop codon occupies the a site no pairing with tRNA is possible. This allows the
release factor to interact with the A site and to promote hydrolysis of the bond linking the
peptide chain to tRNA. In this reaction the release factor causes C-terminal peptiyl
transferase to effect a C-terminal attack by a water molecule instead of by the N-atom of an
incoming tRNA-bound AA. [10]
o A third protein factor, RF3 is involved in the release of RF1/2 from the ribosome. RF3 is a
GTPase. [5]
o After the termination another protein, ribosome recycling factor (RRF) stimulates the
dissociation of the ribosome into subunits thus completing the cycle of protein synthesis. [5]