ribosomal active sites

Question 1

how does puromycin act as a surrogate acceptor site in the A site?

Answer 1

it resembles the 3’ end of the aa-tRNA (CCA terminal)

Question 2

outline the puromycin reaction

Answer 2

puromycin enters the A site of the ribosome, recieving the growing peptide chain, as puromycin has no affinity for the ribosome, the peptidyl-puromycin falls off the ribosome. normally there would be a tRNA attached to the acceptor substrate

Question 3

explain what the works of robin monro found

Answer 3

formed peptide bonds using fmet-tRNA as the donor site and puromycin as the acceptor site, tested this wit both large and small subunits and found that the large ribosomal subunit carries out peptidyl-transferase reactions

Question 4

what observations were made upon cleavage of fmet-tRNA with T1 RNase?

Answer 4

• hexanucleotide carrying fmet was produced, this still worked as a donor substrate but
• required 2’ OH group
• works better when the amino group of the AA is blocked

Question 5

outline peptide bond formation

Answer 5

1- tetrahedral intermediate collapses with the breakage of the bond between the amino group of A site tRNA and carbonyl of P site tRNA
2- the 2’ OH on P site tRNA is involved in proton shuttling to provide a proton after collapse of intermediate

Question 6

what is the structural role of the peptidyl-transferase centre?

Answer 6

two tRNAs come very close together in the ribosome

Question 7

what is the functional role of the peptidyl-transferase centre?

Answer 7

3’ CCA ends of peptidyl-tRNA is held in optimal position by hydrogen bonds from residues on the 23S RNA on the P site and the 23S RNA on the A site, this puts the groups which form a peptide bond in the correct orientation

Question 8

where is the GTPase activation centre located?

Answer 8

on the large subunit of the ribosome

Question 9

what is the ternary complex when referring to the peptidyl transferase centre?

Answer 9

[aa-tRNA-Tu-GTP]

Question 10

which components of the ternary complex are covalently bonded together?

Answer 10

EF-Tu and GTP

Question 11

what is the role of the ribosome with respect to the ternary complex?

Answer 11

scrutinises the ternary complex to decide whether to accept the aa-tRNA that is carried, this is decided by the decoding box

Question 12

which component is responsible for release of GTP from the ternary complex?

Answer 12

as the complex binds to the ribosome, EF-Tu causes the GTP to hydrolyse

Question 13

what is the role of GTP in the ternary complex?

Answer 13

get rid of EF-Tu, which has high affinity for the ribosome. GTP hydrolysis changes the conformational state, causing affinity to decrease.

Question 14

how does GTP binding alter the domain structure of EF-GTP/GDP?

Answer 14

bound GTP allows domain I to move relative to II and III

Question 15

what does the GTP box do?

Answer 15

hydrophobic ‘gates’ consisting of isoleucine and valine are opened and histidine enters the box to hydrolyse GTP

Question 16

what is the role of the amino acid acceptor stem in the ternary complex?

Answer 16

lies across the structure, stabilising it

Question 17

why must the aminoacyl-tRNA be separated from the ternary complex before GTP hydrolysis?

Answer 17

in order to restore flexibility to EF-Tu and generate conditions which permit domain I to move

Question 18

how does the ternary complex fit into the A/A site?

Answer 18

the whole complex cannot, but the anticodon loop can get into the A site on the small subunit, the complex fits into A/Tsite until Tu is lost, and the tRNA can rotate into the A/A site.

Question 19

between which molecules does peptide bond formation take place?

Answer 19

between NH2 of the tRNA in the A site and the carbonyl of the bridging ester bond between the growing peptide and the tRNA in the P site