ALL TRANSLATION BASED MOLECULAR QUESTIONS

1. (a) Briefly describe how the ribosome binding site (RBS) is recognised in prokaryotes during
translation initiation, and explain how this is useful for the translation of polycistronic mRNA.
(6 marks)

Answer:

The mRNA in prokaryotes can code for multiple proetins due to a string of multiple ORF seqeunces.
RBS or ribosome binding site, helps facilitate the expression of these genes because RBS is located
3-9 base pairs upstream from the start codon, and possess 5 bases i.e. GGAGG, located in the SD
seqeunce, which binds to the complimentary base pair on the 16s ribosomal RNA.

This allows the ribosome to be correctly positioned at the start codon of the mRNA (or the ORF,
meaning a string of codons), hence can lead to the expression of multiple genes such as; lactose
metabolism genes. Prokaryotic 16s ribosomes can be assembled wherever there is a Shine-
Dalgarno sequence and bacterial mRNAs can contain more than one start site, hence it is often the
case that one mRNA can code for multiple polypeptides Therefore the mRNA of prokaryotes are
considered to be polycistronic.
(b) Discuss the relevance of the CAP and scan model for understanding the importance of the 5’UTR
in translation regulation in eukaryotes. (12 marks)

Answer:

The 5′ untranslated region (5′ UTR), (also known as a leader sequence or leader RNA) and possess
100-220 nucleotides, and is the region of an mRNA that is directly upstream from the initiation
codon.

The regulation of translation by 5 UTR in eukaryotes is important due to the CAP and scan model,
this is because there are 3 certain mechanisms that can either inhibit or promote translation from
occurring by directly affecting the capability of the CAP and scan model to function.

The first mechanism that has the ability to regulate the rate of translation initiation is via the
stability of the secondary structure in the 5 UTR. This can resist the unwind activity of the Eif4A
helicase, thereby blocking scanning by the pre initiation complex.

This is very effective at inhibiting translation when close to the 5 CAP. More growth factors, and
proto oncogene transcripts have stable secondary structure in the 5 UTR. Overexpression of EiF4F
family of proteins can overcome this inhibition, and can permit the scanning and capping process
to occur as a result.

Another mechanism that can occur is specific RNA binding proteins, these sites for RNA binding
proteins in the 5 UTR will block scanning, hence directly linking toe CAP and scan model and can
thereby inhibit translation.

The secondary structure is often recognised by RNA binding proteins; YB1 binding to stem loop
structure in the 5 UTR of TGF-Beta transcript. DNA mutations can prevent proteins such as many of
the initiation factors required to activate translation.

The final mechanism 5 UTR uses to regulate translation via the CAP and scan model is when
upstream start codons (uAUG or uORF) suppresses the translation of the transcript. DNA
mutations that create spurious upstream AUG will severely inhibit translation. This is due to the
fact that the ribosome isnt binding to the authentic uAUG, this will result in the de-activation of
translation. Without this critical authentic upstream codon, a ribosome pre initiation complex
cannot be formed.

This is because there isn’t any “scanning,” occurring through the 5’UTR, to find the start codon
located within the kozak seqeunce in the first place. However, there are specific DNA mutations,
that can remove pre-existing upstream AUG, to activate AUG, this will permit the ribosome to bind
to the authentic AUG hence initiating the CAP and scan model.
2. Compare and contrast the process of translation initiation in prokaryotes and eukaryotes.
(20 marks)

Answer:

The process of translation initiation between prokaryotes and eukaryotes can be deemed as
similar however strikingly different. For example; The CAP and scan model is involved within
translation initiation in eukaryotes however not prokaryotes.

This process involves the ribosome to be recruited at the 5-cap end, and scans along until an AUG
codon is found, which is referred to as the Kozak seqeunces (G/ANNAUGG) which is where the
start codon is located. A ribosome pre initiation complex is then formed, and has to scan through
the 5’UTR, to find the start codon.

The 5 cap end travels along the 5 to 3 end strands of mRNA, until it reaches the stop codon then
falls off. The ribosome pre initiation complex caused by the 5 UTR happens when, the eIF4F
complex is recruited to the 5′ cap, which in turn recruits the ribosomal complex to the 5′ UTR.
Both eIF4E, eIF4G  eiF4B, and eiF4 A binds to the 5′ UTR, the mRNA associates with initiation
factors before joining ribosome eg eIF4E / G unlike prokaryotes. It is important to note that
eIF4A/B, however is an RNA helicase that removes 2° structure and stimulates ATP-dependent
‘scanning’ for AUG, this does not occur in the initiation of translation for prokaryotes.
Another difference compared to prokaryotes is that, Eukaryotes simply do not have RBS compared
to prokaryotes, this is because Eukaryotes are dependent on the Initiator tRNA to be recruited to
the small ribosome subunit before mRNA binds. This is required since there is no RBS to position
mRNA on ribosome and therefore Positioning is dependent on codon-anti-codon recognition and
therefore requires tRNA (tRNA recognition of flanking Kozak sequence also helps).

Whist Prokaryotes on the other hand use RBS recognition, whereby the RBC or ribosome binding
site, is located 3-9 base pairs upstream from the start codon, and possess 5 bases i.e. GGAGG
which binds to the complimentary base pair on the 16s ribosomal RNA, and allows the ribosome
to be correctly positioned at the start codon of the mRNA, whilst in eukaryotes have only the AUG
start codon on the mRNA, which is vital to initiating the CAP and Scan model to occur via the 5 end
cap required to bind to the start codon via scanning. In addition, Prokaryotes do not need tRNA, to
correctly position the mRNA inorder for correct codon to anticodon base pairing to occur, unlike
eukaryotes.

Prokaryotes also have fewer initiator factor-based proteins to bind to the 5 ends of the mRNA,
whilst there are more initiation factors involved, in eukarytoic initiation i.e eIF4E, eIF4G  eiF4B, and
eiF4A, prokaryotes seem to only utilise IF1,2 and 3. Therefore, there isn’t a pre initiation
translatory complexe that is formed in prokaryotic initiation translation mechanisms only 70S
initiation complexes are formed when Large subunit binding activates GTPase activity of IF2 which
IF2-GDP released, along with IF1.

Furthermore, during RBS recognition, Shine-Dalgarno sequences exist in Prokaryotes which is

On the other hand, eukaryotes use kozak seqeunces, and is different from shine-dalgarno because
Shine-Dalgarno allows bacterial ribosome to be built at an interior position on the mRNA through
direct binding to this sequence.

Another noticeable difference is that The PIC (pre initiation complex) in eukaryotes consists of the
40S ribosomes for eukaryotes that can only recognises one codon, whilst as mentioned the
prokaryotes use 16s ribosomes that bind to the Shine-Dalgarno sequences.

However, in contrast, both systems use translatory based mechanisms that require complimnetray
base pairing, both mechanisms use ribosomes as a platform for both processes to initiate, both
mechanisms use sequences that possess the start codons (AUG) necessary for both processes to
occur.

Other differences include tRNAi which binds to Met not fMet in eukarytoic initiation. Furthermore,
eIF2-GTP ‘escorts’ tRNA’ to the ribosome whilst this is strikingly different to prokaryotes as
mentioned prokaryotes do not utilise tRNA.

On the other hand, similarities to prokaryotes involve a dedicated initiator tRNA, which enters the
P site, as well as the use of initiation factor proteins to block E and A sites on ribosome and most
importantly the use of GTP hydrolysis which acts a driving process that serves as a switch to
convert inactive state initiator factor proteins into an activated state, that allows them to
dissociate from initiator-tRNA.

3. (a) Briefly describe how the ribosome binding site (RBS) is recognised in prokaryotes, and explain
how this is useful for the translation of polycistronic mRNA. (10 marks)
Answer:
The mRNA in prokaryotes can code for multiple proetins due to a string of multiple ORF seqeunces.
RBS or ribosome binding site, helps facilitate the expression of these genes because RBS is located
3-9 base pairs upstream from the start codon, and possess 5 bases i.e. GGAGG, located in the SD
seqeunce, which binds to the complimentary base pair on the 16s ribosomal RNA.

This allows the ribosome to be correctly positioned at the start codon of the mRNA (or the ORF,
meaning a string of codons), hence can lead to the expression of multiple genes such as; lactose
metabolism genes. Prokaryotic 16s ribosomes can be assembled wherever there is a Shine-
Dalgarno sequence and bacterial mRNAs can contain more than one start site, hence it is often the
case that one mRNA can code for multiple polypeptides Therefore the mRNA of prokaryotes are
considered to be polycistronic.

(b) Name two proteins involved in the process of translation that bind GTP, and briefly describe their
role in facilitating this process. (10 marks)

Answer:

There are 2 main proteins that are involved in elongation that bind to GTP, these are by 2
translation elongation factors: EF-Tu, and EF-Ts and is involved in a process called “loading,” and
provides a variety of functions. Firstly, EF-Tu -GTP ‘escorts’ next amino-acyl-tRNA to the ribosome
where it binds the mRNA codon in the A site, AA cannot form a peptide bond because EF-Tu masks
the AA. Only the correct codon-anticodon binding places EF-Tu in factor binding centre and results
in GTP hydrolysis, GTP hydrolysis releases EF-Tu-GDP and unmasks AA, EF-Ts acts as ‘recycling
factor’ for EF-Tu.

4. Discuss the relative importance of RNA and protein during translation elongation, using examples
of specific RNA and protein molecules to illustrate your answer. (20 marks)

Answers:
Elongation consist of 3 main process that enable to drive the entire process these are; Loading,
peptide bond formation and lastly translocation, these 3 steps also possess a spectrum of protein
molecules that enable the facilitation for elongation.

Loading catalysed by 2 translation elongation factors: EF-Tu, and EF-Ts, EF-Tu -GTP ‘escorts’ next
amino-acyl-tRNA to the ribosome where it binds the mRNA codon in the A site, AA cannot form a
peptide bond because EF-Tu masks the AA. Only correct codon-anticodon binding places EF-Tu in
factor binding centre and results in GTP hydrolysis, GTP hydrolysis releases EF-Tu-GDP and
unmasks AA EF-Ts acts as ‘recycling factor’ for EF-Tu.

Peptide bond formation is the next step and involves the tRNA in the P site is released from AA, a
peptide bond forms between the amino acids in A and P sites. Ribosomes keeps the AA-tRNAs in P
and A sites in correct position for peptide bond to form (entropic catalysis), peptidyl transferase
reaction –catalysed by 23S rRNA molecule. P-site tRNA also plays a role -‘substrate-assisted
catalysis. Primarily RNA mediated process; protein components of ribosome seem to have
primarily structural role.

The last step involves translocation, which is defined as the process by which the ribosome moves
one codon along the mRNA, and the tRNAs move into E and P sites, which forms a hybrid state of
tRNA, and occurs where the tRNA is free to move from the adjacent tRNA and overlaps between
the P and A site this is because there is peptide bond formation within the P site originally which
allows the tRNA to move within the P site. This Requires a 3rdelongation factor, EF-G (eEF-2 in
eukaryotes) bound to GTP, EF-G-GTP complex binds to ribosome in A site. Factor binding centre
stimulates GTP hydrolysis and Causes conformation change that triggers translocation, and release
EF-G-GDP.

5. ‘During the termination phase of translation, RNA molecules play a much lesser role than during
initiation or elongation phases. Discuss the validity of this statement, explaining your rationale at
with specific molecular detail.

Answer:
I heavily agree with the statment above as; tRNA in denomination is not involved at all, only
release factor proteins are predominantly utilised in termination. In termination, no tRNA
molecules recognise stop codons there are specific proteins i.e. release factors (RFs) which mimic
the tRNA and anti-codon structure, they catalyse the release of polypeptide from peptidyl tRNA.
There are generally two classes of RFs: class I and class II, class I RFs recognise stop codon and
catalyse hydrolysis and release of polypeptide chain whilst class II RFs and trigger release of class I
RFs. class II RFs are GTPases.

Another example of proteins involved in termination that are not tRNA are part of a process
involved in prokaryotes where ribosomal recycling is facilitated these are by RRF, in combination
with EF-G and IF3. RRF binds A-site (tRNA mimic) and recruits EF-G-GTP. EF-G-GTP hydrolysis in the
presence of RRF causes release of both P-site and E-site tRNAs.IF3 facilitates both release of mRNA
and ribosome subunit dissociation.

6. In which of the 3 stages of translation (initiation, elongation and termination) would you say that
RNA plays a dominant role in facilitating the process, and in which stage would you say that proteins
plays a dominant role. Justify/explain your answer.

Answer:
Initiation is the process that involves predominantly RNA, this is because RNA more specially
mRNA is a major component required for the onset of translation, for example for eukaryotes
ribosomes must be recruited at the 5-cap end of the mRNA, and scans along until an AUG codon is
found on the mRNA, which is referred to as the Kozak seqeunces (G/ANNAUGG) which is where
the start codon is located. A ribosome pre initiation complex is then formed, and has to scan
through the 5’UTR, to find the start codon.

Elongation is the process by which proteins play a predominant role for this process to be driven.
Elongation consist of 3 main process that enable to drive the entire process these are; Loading,
peptide bond formation and lastly translocation, these 3 steps also possess a spectrum of protein
molecules that enable the facilitation for elongation.

Loading catalysed by 2 translation elongation factors: EF-Tu, and EF-Ts, EF-Tu -GTP ‘escorts’ next
amino-acyl-tRNA to the ribosome where it binds the mRNA codon in the A site, AA cannot form a
peptide bond because EF-Tu masks the AA. Only correct codon-anticodon binding places EF-Tu in
factor binding centre and results in GTP hydrolysis, GTP hydrolysis releases EF-Tu-GDP and
unmasks AA EF-Ts acts as ‘recycling factor’ for EF-Tu.

Peptide bond formation is the next step and is heavily reliant on proetins involves the tRNA in the
P site is released from AA, a peptide bond forms between the amino acids in A and P sites.
Ribosomes keeps the AA-tRNAs in P and A sites in correct position for peptide bond to form
(entropic catalysis), peptidyl transferase reaction –catalysed by 23S rRNA molecule. P-site tRNA
also plays a role -‘substrate-assisted catalysis. Primarily RNA mediated process; protein
components of ribosome seem to have primarily structural role.

The last step involves translocation, which is defined as the process by which the ribosome moves
one codon along the mRNA, and the tRNAs move into E and P sites, which forms a hybrid state of
tRNA, and occurs where the tRNA is free to move from the adjacent tRNA and overlaps between
the P and A site this is because there is peptide bond formation within the P site originally which
allows the tRNA to move within the P site. This Requires a 3rdelongation factor, EF-G (eEF-2 in
eukaryotes) bound to GTP, EF-G-GTP complex binds to ribosome in A site. Factor binding centre
stimulates GTP hydrolysis and Causes conformation change that triggers translocation, and release
EF-G-GDP.

7. List the GTP-binding proteins that are involved in prokaryotic translation. For each protein write a
sentence to describe it’s function.

Answer:

In initiation of translation for eukaryotes on main GTP binding protein is eIF2-GTP, and the main
function is to ‘escorts’ tRNA’ to the ribosome. In prokaryotes the Large subunit binding activates
GTPase activity of IF2 which causes IF2-GDP to be released, along with IF1 forming the 70S
initiation complex.
There are 2 main proteins that are involved in elongation that bind to GTP, these are by 2
translation elongation factors: EF-Tu, and EF-Ts and is involved in a process called “loading,” and
provides a variety of functions. Firstly, EF-Tu -GTP ‘escorts’ next amino-acyl-tRNA to the ribosome
where it binds the mRNA codon in the A site, AA cannot form a peptide bond because EF-Tu masks
the AA. Only the correct codon-anticodon binding places EF-Tu in factor binding centre and results
in GTP hydrolysis, GTP hydrolysis releases EF-Tu-GDP and unmasks AA, EF-Ts acts as ‘recycling
factor’ for EF-Tu.

In termination Class II release factors are GTP-binding proteins that serve to enhance the efficiency
of translation termination, either by promoting release of the Class I factor from the ribosome
after peptidyl release (as in the case of bacterial release factors) or by forming a complex with
the Class I RF prior to stop codon.

Another example of GTP proteins involved in termination is EF-G-GTP. EF-G-GTP hydrolysis in the
presence of RRF causes release of both P-site and E-site tRNAs.IF3 facilitates both release of mRNA
and ribosome subunit dissociation.