LEC26: Translation Machinery & Mechanism I

Question 1

what is translation?

what codes for what?

Answer 1

converting nucleic acid info into protein info

mRNA codon codes for nucleic acid

Question 2

what is a tRNA?

what does it do?

Answer 2

transfer RNA

recognizes an mRNA codon via its own complementary anticodon

produces corresponding **amino acid **at its 3’ end

Question 3

what holds together the tRNA structure?

Answer 3

intramolecular base pairing

Question 4

what is the structure of tRNA overall?

Answer 4

3’ end CCA codon, A attaches via OH to alpha-carboxyl group of attached amino acid, covalent link

T loop, anticodon loop, D loop

anticodon that’s complementary to codon

5’ end

Question 5

do some amino acids have >1 tRNA to whicih they can be attached?

Answer 5

yes

Question 6

what do aminoacyl tRNA synthetases do?

Answer 6

covalently couple each amino acid to its appropriate set of tRNA molecules; diff synthetase enzyme for each a.a.

proofreading

taks a.a. and activates teh a.a.

must be able to discriminate vs. diff. a.a., which come into its active site

thus has **proofreading function **so can recognize if it has put the wrong a.a. in place, so can go back, hydrolyze it off the tRNA

Question 7

what is wobble pairing?

Answer 7

the last ntd in a codon and 1st ntd in anticodon don’t have to be classic base pairing

not every codon requires a dedicated tRNA w/ standard codon:anticodon base pairing, can have variability in that position

Question 8

what are the machines of translation?

Answer 8

mRNA, tRNA, ribosome

Question 9

what do aminoacyl tRNA synthesases do?

Answer 9

enzymes

catalyze covalent attachment of a cognate a.a., based mainly on anticodon sequence, to CCA 3’ end of tRNA

Question 10

READ tRNA SYNTHETASE-catalyzed RXN

Answer 10

attaches the amino acid to the 3’ end of the tRNA

an energy-releasing hydrolysis of ATP

produces high-energy bond btwn charged tRNA and the a.a.

this bond’s energy is used later in protein synthesis, links amino acid covalently to growing peptide chain

Question 11

what is the structure of the ribosome?

what does each part do during translation?

Answer 11

large subunit = 60S; small subunit= 40S, together make **80S, **contain **proteins & rRNA **

small subunit: matches tRNAs to codons of mRNA

large subunit: catalyzes formation of peptide bonds that covalently link a.a. together into polypeptide chain

Question 12

what does rRNA do in the ribosome?

Answer 12

does catalytic fxn of ribosome - makes a peptide bond

therefore ribosome is a ribozyme

Question 13

what is the structure of the rRNA of the ribosome?

Answer 13

18S unit, 28S unit

Question 14

what is this?

Answer 14

18S rRNA of the small (40S) subunit of the ribosome

Question 15

what are the 3 “sites” on the ribosome for tRNA?

Answer 15

E=exit site; P=peptidyl site; A=aminoacyl site

imagined sites where tRNA bond, do different stages of translation

Question 16

which part of the tRNA binds to which part of the ribosome during translation?

Answer 16

bottom part of tRNA w/ anticodon is in 40S subunit of ribosome

top part of tRNA w/ amino acid binds to 60S subunit of ribosome

Question 17

what happens in each “tRNA site” on ribosome?

Answer 17

A site: wher enext charged tRNA binds

P site: where tRNA w/ growing peptide chain is located

E site: where tRNA in P site goes after its peptide cargo has engaged in peptide bond synthesis

Question 18

what are eIFs? what do they do?

Answer 18

eukaryotic initiation factors

bind ribsome to mRNA 5’ end, position ribosome for correct translation initiation

Question 19

what are eEFs? fxn?

Answer 19

eukaryotic elongation factors (have 3!)

bring succeeding charged tRNAs to the site on ribosome where peptide bond formation occurs

also translocate ribosome to next codon

Question 20

what is eRF? fxn?

Answer 20

eukaryotic release factor

just have 1!

relases finished polypeptide upon encountering stop codon, releases ribosome from the mRNA

Question 21

where does the first tRNA enter the ribosome? explain uniqueness of 1st tRNA

Answer 21

1st tRNA for AUG codon (Met) enters P** site; every subsequent tRNA enters into A site**

**1st tRNA = tRNAi, initiation tRNA for Met **

have another tRNA for Met within the chain

Question 22

what is the preinitiation complex of translation made of?

Answer 22

Met tRNAi in its GTP-bound state

also have 40S subunit, eIFs

so

ternary complex of Met-tRNAi-EIF2-GTP w/ 40S ribosome subunit, eIFs

Question 23

what is the eIF4 complex of tln initiation factors?

Answer 23

form 5’-cap binding complex

recognizes the cap specifically

brings w/ it all the other eIF4s that make this complex of eIFS that’re bound to mRNA

Question 24

what does the 40S ribisome subunit do re: initiation of tln?

Answer 24

part of PIC

binds the 5’ end of the mRNA

at expense of ATP hydrolysis, “scans” in 5’ to 3’ direction for an AUG that’s in correct sequence context to fxn as start codon

Question 25

what is the process of translation kozak scanning?

Answer 25

must find start codon, which is internal, after the 5’UTR

find it by** kozak scanning model**: 40S of the PIC complex scans via ATP hydrolysis in 5’ to 3’ direction for an AUG start codon

Question 26

how does full ribosome get positioned for tln?

Answer 26

EIF2 departs from PIC, via hydrolysis of GTP to GDP

however, EIF2 isn’t a good phosphatase; needs a GAP to work

**EI5 is EIF2’s GAP that hydrolyzes GTP to GDP **

NOW 60S subunit binds, 80S ribosome is formed

Question 27

what happens to EIF2 after it’s removed from PIC?

Answer 27

returns to its GTP-bound state via aid of **GEF: eIF2B, **which **catalyzes exchange of GDP for GTP **so now **EIF2 **can pick up next **Met-tRNAi **for next ronud of tln

Question 28

what is vanishing white matter disease?

Answer 28

an eIF2B defiency (the EIF2 GEF that restores GTP-bound state for EIF2)