Enzymology - Week 6

Question 1

what happens during initiation, elongation and termination in transcription briefly

Answer 1

Translation occurs by:
- Initiation
> mRNA and tRNA are charged with the first amino acid and the 2 ribosomal subunits assemble into a complex to form the initiation complex.
- Elongation
> Amino acids are added one by one to a growing polypeptide.
- Termination
> Polypeptide chain is released from tRNA, and ribosome dissociates from mRNA.

Question 2

how does tRNA gain its modified amino acid

Answer 2

The initiator tRNA gains its modified amino acid in a two-stage reaction:
1. it is charged with the amino acid to generate Met-tRNAf
2. the formylation reaction blocks the free NH2 group, which prevents the initiator from participating in chain elongation

Question 3

where does translation start in eukaryotes and in bacteria

Answer 3

In bacteria, the first codon in the mRNA (AUG) leads to the initiation and recruitment of the formyl-methionyl tRNA.
Unmodified methionines initiate translation in eukaryotes

The modification of the methionine only happens in bacteria and not in eukaryotes. Thus we can say that translation starts at the same amino acid, that is the methionine in eukaryotes but not in bacteria as the methionine is modified in bacteria.

Question 4

what happens during the initation stage in bacteria and what is needed in the initiation stage in bacteria

Answer 4

Initiation of translation in bacteria requires:
- An mRNA
- A ribosome
- A specific initiator tRNA
- Three initiation factors (IFs)

Question 5

what is the complex made up of in the initiation stage in bacteria and what is required to help make the complex

Answer 5

During the translation stage, a complex is formed between the mRNA molecule, the first tRNA and the ribosomal subunit.
Also, the formation of this complex requires the help of proteins that are called initiation factors (IFs).

Question 6

how is the initiation complex formed in brief

Answer 6

Initiation complex formation occurs in three steps:
- Binding of 3 IFs and GTP to the small ribosomal subunit.
-Binding of fMet-tRNA (initiator aminoacyl tRNA) to the 30S subunit and mRNA. IF1 occludes the A site. IF2 binds the initiator and helps it to attach to the small ribosomal subunit
-IF3 must be released from the complex in order to enable the 50S subunit to join
-IFs are released and the large ribosomal subunit joins the complex. The GTP is hydrolyzed when the 50S subunit joins to generate a complete ribosome.

Question 7

what happens in the first 2 steps in DETAIL of the initiation complex formation

Answer 7

First the 3 initiation factors 1, 2 and 3 bind to the small subunit of the ribosome.

Then the initiator tRNA, in this case, formyl-methionine tRNA and the mRNA join the complex, the mRNA contains a specific sequence called the Shine-Dalgarno sequence which is used as the binding site of the ribosome.

Then in the P-site of the ribosome, the initiator factor 1 (FI1) prevents the tRNA initiator to bind to the mRNA in another location of the ribosome. For example in the A-site, because the IF1 occupies the A-site. So essentially the initiator tRNA binds to mRNA in the P-site of the ribosome.

Question 8

what happens in the last step in detail of the formation of initation complex and what is the outcome of the initiation stage

Answer 8

Then the initiation factor 3 (IF3) is released from the complex and the largest subunit can join the complex. During this step, there is the hydrolysis of GTP that is transformed into GDP. Thus, the initiation stage requires energy in form of GTP. The hydrolyzation of GTP is catalysed by the initiation factor 2 (IF2).

The outcome of the initiation stage is the formation of the initiation complex which consists of the ribosome, mRNA and the first tRNA in the P-site of the ribosome.

Question 9

what is Shine-Dalgarno sequence, where is it found and why is it important

Answer 9

The Shine-Dalgarno sequence is a consensus sequence in the mRNA. It is located a few nucleotides upstream of the start codon.

The Shine-Dalgarno sequence is complementary to a short sequence of the 16S rRNA. It forms a hydrogen bond with the 16S ribosomal RNA (rRNA) within the ribosome. This is important because the ribosome is the recognition site of the ribosome to find the first codon, which is the start codon.

This Shine-Dalgarno sequence is only found in bacteria.

Question 10

what are the 3 elongation factors that are used in the elongation stage of translation

Answer 10

The next stage of translation after initiation is the elongation stage. During the elongation stage of translation amino acids are added one at a time to the growing polypeptide.

This stage also requires the help of proteins called Elongation factors. In bacteria there are 3 elongation factors that are used are:
- EF-Tu
- EF-Ts
- EF-G

(EF stands for elongation factors)

Question 11

What happens in the elongation step in DETAIL in translation

Answer 11

The elongation step of translation can be divided into 3 steps:
1- There is the binding of the aminoacyl tRNA, essentially a charged tRNA is delivered to the A-site of the ribosome by the elongation factor EF-Tu.
2- The second step of the elongation stage is peptide bond formation.
3- After the peptide bond formation is the 3rd step which is called translocation.

Question 12

what happens in the first step in DETAIL in elongation of translation

Answer 12

1- There is the binding of the aminoacyl tRNA, essentially a charged tRNA is delivered to the A-site of the ribosome by the elongation factor EF-Tu.
During the elongation stage of translation the charged tRNA binds to the ribosome at the A-site because the initiator tRNA binds to the ribosome of the mRNA at the P-site.
There is the hydrolysis of the GTP bound to the EF-Tu.
Then the EF-Tu is re-charged by another elongation factor that is called EF-Ts.

Question 13

what happens in the second step in DETAIL in the elongation of translation

Answer 13

2- The second step of the elongation stage is peptide bond formation.
A peptidyl-transferase forms a peptide bond between the amino acid attached to the tRNA in the P-site and the amino acid attached to the tRNA in the A-site.
It is an enzymatic reaction. This enzymatic reaction is made by a large subunit of the ribosome. Thus the ribosome has a catalytic activity, thus it can be said that the ribosome is essentially a Ribozyme ( which is a ribonucleic acid enzyme).

Question 14

what happens in the third step in DETAIL in elongation of translation

Answer 14

3- After the peptide bond formation is the 3rd step which is called translocation.
The 3rd elongation factor which is EF-G pushes the tRNA with the attached polypeptide from the A-site to the P-site. This movement causes it to take the mRNA with it.
As a consequence, the tRNA moves from the P-site to the E-site, and there is the release of empty tRNA.

Question 15

what is the outcome of the elongation stage in translation

Answer 15

All these events, the binding of the charged tRNA, the polypeptide-bond formation and translocation are repeated for each additional amino acid.

Thus the outcome of the translocation is that the next mRNA codon is moved into the A-site, thus it is exposed to the next elongation cycle.

Question 16

what are the 3 release factors in bacteria

Answer 16

In bacteria there are 3 release factors:
-RF-1
-RF-2
-RF-3

Question 17

when does elongation stop in translation and what happens at the termination step

Answer 17

The elongation step continues until a stop codon within the mRNA moves to the A-site of the ribosome.
In bacteria, there are 3 specific stop codons so when one of the stop codons moves into the A-site of the ribosome. It is recognised by a specific release factor. this is when termination occurs.

when the stop codon at the A-site is recognised by a release factor, the peptide-bond formation cannot occur and thus there is a reaction between the polypeptide and water.

when the polypeptide is released into the cytosol, a free tRNA is released into the cytosol and the 2 ribosomal subunits are again separated and released (disassociated) from the complex.

Question 18

what happen after the polypeptide molecule is free after the termination step

Answer 18

When the polypeptide molecule is free in the cytosol and it may be subject to further chemical modifications to form a functional polypeptide.

Question 19

what are the differences in translation between bacteria and eukaryotes

Answer 19

there are some differences in eukaryotes compared to bacteria, Which are:
- Ribosomes are slightly larger in eukaryotes
- The initiator tRNA is methionine but it is not formulated in eukaryotes
- The start codon is always AUG in eukaryotes.
- There is no Shine-Dalgarno sequence in eukaryotes and also bacterial mRNA lacks the cap at the 5’-end and the poly(A)-tail at the 3’-end whereas in eukaryotes mRNA contains a ‘Cap’ at the 5’-end and poly(A)-tail at the 3’-end and there is no Shine-Dalgardo sequence.
- The mRNA contains only 1 open reading frame (ORF). Essentially the mRNA is able to form 1 single polypeptide. By contrast, bacterial mRNA contains multiple ORFs so 1 mRNA is able to produce different proteins thus the mRNA in bacteria is also called poly-cystronic

Question 20

what causes the differences between bacteria; and eukaryotic translation

Answer 20

A difference between bacterial translation and eukaryotic translation is related to the translation factors.

• 3 translation factors are required in the initiation stage of bacteria but many initiation factors are required in the initiation stage of translation in eukaryotes.
• 3 elongation factors are required in both bacteria and eukaryotes.
• Another difference is 3 release factors are required in the termination stage in bacteria but 2 release factors are required in eukaryotes.

Question 21

how is the initiation complex formed/ initiation step carried out in eukaryotic translation

Answer 21

Step 1 - Initiation stage of Translation in eukaryotes
The first step in initiation is when elF2 binds to the initiator tRNA, which is the methionine that is not modified to form a group. The initiator tRNA and the elF2 bind to the small ribosomal subunit.

Step 2 - Then there is the initiator factor elF4E recognises the cap at the 5’-end of the mRNA and the polyA tail at the 3’-end of the mRNA. The recognition is followed by the formation of a closed loop in which the elF4 binds the 5’-end and the 3’-end of the mRNA.

Step 3 - Then the complex ( which is the initiator tRNA and the small ribosomal subunit) moves along the RNA searching for the start codon because there is no Shine-Dalgardo sequence. Here the complex scans the mRNA for the start codon and stops there.

Step 4 - Then the large ribosomal subunit binds to the complex forming the initiation complex.

Question 22

what happens in the elongation stage in translation of eukaryotes

Answer 22

The elongation step of translation in eukaryotes is similar to that in bacteria.
Each charged tRNA is delivered to the 80S ribosome A site by eEF1A·GTP.
Following ribosome-catalysed peptide bond formation, eukaryotic elongation factor 2 (eEF2) catalyses 80S translocation, transferring the deacetylated tRNA to the E site, positioning the peptidyl-tRNA in the P site and re-exposing the A site.

in short, The elongation stage can be divided into the following:
- the entry of a charged tRNA
- the peptide bond formation
- translocation

Question 23

what happens at the termination stage in translation of eukaryotes

Answer 23

The termination stage of translation in eukaryotes is similar to the bacterial termination stage.

Eukaryotic release factor 1 (eRF1) recognizes the stop codon in the A site, triggering 80S arrest and polypeptide release.
eRF3 releases eRF1 from the ribosome, and several initiation factors, together with ABCE1-directed nucleotide hydrolysis, dismantle the complex, thus recycling ribosome subunits.