Translation 4- Elongation

Question 1

What are the 3 steps in elongation

Answer 1

1. Decoding
2. Transpeptidation
3. Translocation

Question 2

What happens in decoding

Answer 2

1. The ribosome selects and binds an aminoacyl-tRNA whose anticodon is complementary to the mRNA codon in the A site
2. Decoding factors

Question 3

What happens in transpeptidation (peptidyl transferase)

Answer 3

1. the peptidyl group on the P-site tRNA is transferred to the aminoacyl group in the A site through the formation of a peptide bond
2. Catalysis of peptide bond formation between the amino acid on new tRNA and formylated methionyl

Question 4

What happens in translocation

Answer 4

1. A-site and P-site tRNAs are respectively transferred to the P site and E site accompanied by their bound mRNA
2. The mRNA together with its base paired tRNAs is ratcheted through the ribosome by one codon

Question 5

What are the elongation factors

Answer 5

1. EF-Tu- binds aminoacyl-tRNA and GTP
2. EF-Ts- Displaces GDP from EF-Tu
3. EF-G- Promotes translocation through GTP binding and hydrolysis

Question 6

What is needed for decoding

Answer 6

1. Binding of aminoacyl-tRNA to the A site
2. Elongation factor EF-Tu- delivers tRNA
3. GTP- normally GTP hydrolysis to GDP is slow, in presence of a gap this hydrolysis is fast
4. Elongation factor EF-Ts

Question 7

What is structure of EF-TU

Answer 7

1. Replaced GTP with non-hydrolysable mimic GDPNP- so it not hydrolysed before structure is taken in x-ray crystallography
2. 3 domains
3. GTP/GDP binding domain
4. Domain 2- binds to aminoacyl site-
5. Domain 3- beta barrel domain – binds to T loop
6. 2 and 3 are tRNA binding domain

Question 8

Why doesn’t the initiator tRNA never read internal AUG or GUG codons

Answer 8

1. EF-TU binds neither formylated aminoacyl-tRNA nor unformylated Met-tRNAfMet that’s why the initiator tRNA never reads internal AUG or GUG codons.
2. The first basepair is mismatched and therefore has a 3’ overhang of 5 nucleotides.
3. This mismatch and the formyl group prevent this binding

Question 9

What happens in decoding

Answer 9

1. A binary complex of GTP with EF-Tu combines with an aminoacyl-tRNA
2. The resulting ternary complex binds to the ribosome and in GTP hydrolysis reaction, the aminoacyl-tRNA is bound in a codon-anticodon complex to the A site and EF-Tu
3. The bound GDP is replaced by GTP mediated by EF-Ts

Question 10

What do EF-T and EF-Tu act as

Answer 10

1. EF-Ts- GEF
2. EF-Tu- GAP

Question 11

Where do EF-Tu and tRNA bind

Answer 11

1. One domain binds to TpsiC arm
2. Other binds to acceptor stem
3. Recognise any tRNA
4. All depends on hydrolysis of GTP
5. Needs to be close to ribosome, then hydrolysed then get conformational change

Question 12

What happens to EF-Tu when GTP is hydrolysed

Answer 12

1. Undergoes a major conformational change on hydrolysing its bound GTP
2. The switch I regions of domain 1 converts from a beta hairpin to a short alpha helix
3. The alpha helix of Switch II shifts toward the C-terminus by 4 residues, therefore the helix reorients by 42 degrees which results results in domain 1 changing its orientation with respect to the other domain (idk if two or 3 domains altogether)
4. This eliminates the tRNA binding site

Question 13

What does EF-Ts do

Answer 13

1. Disrupts binding of GDP to EF-Tu
2. EF-Tu has higher affinity for GDP than GTP so replacement of GDP by GTP is facilitated by EF-Ts interaction with EF-Tu
3. It binds along right side of EF-Tu and its side chains interact with the GDP binding pocket on EF-Tu domain 1- reducing affinity for GDP
4. Binds to EF-TU to activate the EF-TU after it has been deactivated by hydrolysis and undergone a conformational change

Question 14

What happens in transpeptidation

Answer 14

1. Nucleophilic displacement of the P-site tRNA by the amino group of the 3’-linked aminoacyl-tRNA in the A site
2. The nascent chain is thereby lengthened at its C-terminus by one residue and transferred to the A-site tRNA
3. Reaction occurs without the need of activating cofactors suhc as ATP as the ester linkage between the nascent polypeptide and the P-site tRNA is a high energy bond
   catalysed by ribosome – proton abstraction facilitates nucleophilic attack

Question 15

What catalyses transpeptidation

Answer 15

1. catalysed by ribosome – proton abstraction facilitates nucleophilic attack
2. Peptidyl transferase centre located entirely on the large subunit

Question 16

What does translocation require

Answer 16

1. EF-G

Question 17

What does EF-G do

Answer 17

1. Binds to the ribosome together with GTP and its only released on hydrolysis of the GTP to GDP + Pi
2. EF-G release is a prerequisite for beginning the next elongation cycle as EF-G and EF-Tu bind to the same site of the ribosome and hence their binding is mutually exclusive
3. Initially EF-G is compact upon hydrolysis it becomes elongated

Question 18

What happens in translocation

Answer 18

1. The now uncharged P-site tRNA is transferred to the E site, its former occupant having been previously expelled
2. Simultaneously the peptidyl-tRNA in the A site, together with its bound mRNA, is moved to the P site

Question 19

Describe the mimicry of EF-G

Answer 19

1. EF-Tu looks very similar to EF-G
2. But EF-G is all made of protein
3. Seen also in eukaryotic elongation
4. First two domains closely resemble those in EF-Tu.GMPPNP
5. The three EF-G C-terminal domains which have no counterparts in EF-Tu closely resemble the EF-Tu bound tRNA in shape