Translation

Question 1

Relate the structure of tRNA to its function

Answer 1

tRNAs mediate the interaction of amino acids with the genetic code.

They have a universal clover leaf structure; final structure maintained by tertiary hydrogen bonds.

tRNAs become linked to ONE aa (covalently) and brings it to ribosomes in respond to a SPECIFIC codon. They recognise multiple codons each.

Each tRNA has a triplet anticodon that recognises one or more codons in the mRNA by base pairing. (common features)

specific features needed for charging.

It is modified post transcription with varying complexity.

Question 2

the process of tRNA charging

Answer 2

This is a two step process

i) aa +ATP = aa-AMP + PPi

amino acid is ccovalently cleaved to AMP using ATP as a donor; the bonds maintain this high energy as a result. PPi drives reaction

ii) aa-AMP +tRNA = aa-tRNA +AMP

AMP is released

Question 3

how is charging accuracy controlled?

Answer 3

It is important as amino acid doesnt have a role in recognition itself once it has bound but the tRNA charge does.

The discriminator base in the tRNA structure controls identity.

1) tRNA synthetases recognises by contact on the receptor stem which align on one side.
2) change of ONE base on the acceptor change identity ie at the discriminator.
3) proofreading of both aa and tRNA selection.

Question 4

How does codon and anticodon (on tRNA) work?

Answer 4

base pairs between codon and anticodon result in DECODING.

The flexibily of the anticodon loop meand base pair rules are relaxed; U could pair with A or G! (WOBBLES)

This allows tRNA to bind to more than one codon and decode a CODON PAIR.

unique codons must end in G or U because A and C cannot have a unique meaning

Question 5

Why is the genetic code organised in a certain way?

Answer 5

The Genetic Code consists of NON-overlapping triplets which are read from a fixed starting point (we know this because single base mutaions in gene cause single aa defects.)

There are different possible codons (so code is degenerative); proportional to the size of the specific protein.

61 codons (sense) and 3 stop codons.

Question 6

structure in relation to current models of how ribosomes function

Answer 6

Ribosomes 70S made of 50S and 30S of a combination of rRna and proteins.

Recognition of the correct start (AUG) codon requires the 3’-end of 16S rRNA on the 30S component.

50s Primarily RNA which therefore determines shape and function of ribosome; proteins (most non essential) fill the gaps

TRNA BIDING DIRECTLY INVOLVES rRNA! binding will change rRNA conformation

X-ray crystallographic structures deduced from Chemical crosslinking and
Conservation of base pairing potential in many bacterial rRNA sequences

Question 7

How is the overall accuracy of protein synthesis maintained?

Answer 7

There is a very low error rate for charging or synthesis. this is due to Proof reading; correct codon-anticodon interaction triggers GTP hydrolysis (EF-Tu) and stable aa~tRNA binding to the ribosome.

If not completely correct fit shows differing kinetics ; ribosome bases monitor the codon-anticodon in the minor groove

There are 2 PHASES TO PROOFREADING

i) ternary complex of aa-RNA binding
ii) cognate aa after factors disassociate.

When correct: tRNA transmits a signal from decoding center so GTP hydrolysis increases and aa-t-RNA is relaxed from bent EF-Tu-GTP form to its normal ACCOMMODATED form.

Question 8

What is the difference between prokaryotes and eukaryotes?

Answer 8

1) bigger ribosome, different sructure and initation
2) different mRNA; prokaryoic is polycistronic mRNA (unstable), eukaryotic is monocisronic (stable)
3) Simultaneous transcription and translation in prokaryotes; separtion of processes in eurkayotes

Question 9

Translational INITATION.

Answer 9

Occurs at AUG (methionine) codons

fmet-tRNA is initiator which is made after charging, entering the P SITE due to specific properties in the anticodon stem; ever other tRNA mus enter from A site to add more amino acids to chain held in P site.

Met-tRNA does the rest

The binding site is PURINE RICH (A OR G)

Question 10

How is Eurkaryotes translational INITATION regulated?

Answer 10

Euryotic ribosome made up of 40S and 60s of varied RNA and proteins. There are two elongation factors (EF-TU = EF1a; EF-G=EF2)

There is NO BASE pairing interaction of a Ribosome Binding Site (RBS) with the 18S rRNA, instead Ribosomes bind to the 5’ end of the mRNA and scan for the first AUG codon
5’ end of mRNA is recognised by a special ‘cap’ that is added post-transcriptionally

The initator tRNA is NOT formylated

An assembly of complexes is needed to initiate that if bounf to elF4 stops at the 43S stage and mrna codon translation will not continue

Question 11

What direction is the mRNA read?

Answer 11

5’ to 3’ and the protein as made from the N to C

Question 12

What are the reasons for MODIFYING tRNA?

Answer 12

To make the binding of different aminoacyl-tRNAs to both the A and P sites of the ribosome UNIFORM, otherwise its sequence would affect the binding affinity for the two principal tRNA binding sites on the ribosome

Anticodon modifications can affect the nature and efficiency of base-pairing with codons (used in decoding)

Question 13

What is meant by cognate amino acid?

Answer 13

as specified by the anticodon; tRNAs bind to cognate aa via the enzyme aminoacyl-tRNA synthetase making an ester bond on the OH group

Question 14

Describe proofreading

Answer 14

if the wrong component has been selected it either DISFAVORS the forward reaction (kinetic) or REVERSES the catalytic reaction (chemical proofreading) There are two active sites: active site and an editing site; the aa has to fit with BOTH.

It occurs when aa binds the tRNA; if mischarged it is hydrolysed. Ile and Val very similar and tRNA could accept the wrong aa for the codon.

If correct bind occurs there is a conformation change in the acceptor stem moves the aa to the EDITING SITE (this doesnt happen in incorrect binding so there is time here for tRNA to dissociate)

Question 15

Why is accuracy maintained

Answer 15

STICKY mutation of the editing site, means proof reading doesnt work so tRNA is allowed to be mischarged and then develops to a neurodegenerative condition

Question 16

How does modification of base pairs in wobble occur?

Answer 16

1) Inosine (I) pairs with A, U or C (e.g. tRNAile) he sulfur prevents G from binding. This means AUA is read but AUG cannot be.
2) 2-thiouridine only pairs with A and not G

Question 17

Basic wobble rules

Answer 17

Wobble rules define a minimal set of 31 tRNAs plus an initiator tRNA for translating the universal genetic code

Question 18

History of ribosome structure

Answer 18

early: Overall shape deduced by electron microscopy and antibodies, neutron scattering found the proteins

Question 19

the role of the T, A, P and E tRNA binding sites on the ribosome

Answer 19

A- where tRNA binds in response to next codon

P- carries tRNA along growing peptide.

Translocation resets the ribosome by moving new tRNA back to P site as the ribosome moves along one base codon.

The E site (Exit site) is where the deacylated tRNA is placed before it leaves the ribosome

The T site (Tu site) is to be the initial site of EF-Tu interaction

Question 20

Describe the Ribosome Binding site properites

Answer 20

Purine-rich sequence, 8-13 bases before the start codon
Subset of -AGGAGG- such as -GAGG- or -GGAG-
Base-pairs with the 3’ end of 16S rRNA
Importance deduced from experiments with bacteriophage T7 gene 0.3

Question 21

NAME Initation factors and what they do

Answer 21

Factors are on NON TRANSLATING 30S component enhancing initation
1) IF1 recycles IF2/3.

2) IF2: binds GTP and initiator tRNA (fmet-tRNAmetf) and places the starting complex into P- SITE. Once bound GTP hydrolyses and IF2 is released.
3) IF3: prevents reassociation of 30S and 50S subunits so the 30S subunit can bind to mRNA

50S is then added as factors are hydrolysed by GTP and expelled

Question 22

Elongation

Answer 22

SUMMARISED:
- initated in P site before elgonation.

-Aminoacyl-tRNA entry into A site as a ternary (3 components). An elongation factor helps here which is hydrolysed by GTPonce it binds correctly.

-Peptide bond synthesis
once EF-Tu-GTP complex is released. This is in the 23S component of the 50S subunit

-Translocation

Question 23

What does peptidyl transferase do? How Is it a RIBOZYME?

Answer 23

When the correct tRNA are in the P and A site it makes a new PEPTIDE BOND between the GROWING chain on the P site and the amino acid of the Asite in an ESTER-AMINE INTERACTION. This is a spontaneous reaction, (even faster in ribosome) where the ribosome sheilds reaction from solvent

BLOCKED by: Chloramphenicol (bacteria)
Anisomycin (eukaryotes)

Without proteins peptide bond still occurs, with proteins reaction activity is enhanced.

Question 24

What happens in TRANSLOCATION?

Answer 24

The ribosome is reset

the deacylated tRNA is ejected from the P site.

the peptidyl-tRNA in the A site is moved to the P-site together with its codon.

The next codon enters the A-site

Translocation is Mediated by EF-G and GTP;

i) EFG-GTP binds to A site, allowing translocation
ii) hydrolysis of GTP expels the factor

Sparsomycin triggers translocation in the absence of EF-G•GTP

EF-G and tRNAEF-TU are very similar and explains how the have same binding site (mimic)

Question 25

What recognizes STOP codons?

Answer 25

release factors; different factors (RF1-3, RRF) recognize different codons

These act on the A site wih a paptidyl presnt in the P site.

Question 26

What are the functions of a release factor?

Answer 26

RF1 recognises UAA and UAG

RF2 recognises UAA and UGA (mimic tRNA)

RF3•GTP promotes hydrolysis of the completed peptide
Expulsion of the deacylated tRNA (mimics the GTP-binding domain of EF-Tu and EF-G)

Allows RF3 to position a water molecule to which the polypeptidyl is transferred instead of to an aminoacyl-tRNA

RRF (ribosome releasing factor) promotes dissociation of the ribosomal subunits, tRNA and mRNA

Question 27

What happens with stalled ribosomes?

Answer 27

there is a vacant Asite; the end of the mRNA has been reached before a stop codon, if i was damaged.

To free ribosomes: TmRNA releases stalled ribosome and taggs released peptide with the sequence TmORF which is recognised by proteases and degraded

Question 28

What is TmRNA?

Answer 28

an RNA that folds up like a t-RNA structure and can be CHARGED WITH ALANINE.

It has a central peptide.

Question 29

What relevance does the Hybrid sites model have?

Answer 29

Extends our understanding of the basic mechanisms

Now we know, two more binding sites E (EXIT) site and T (TU) site

Question 30

Describe hybrid binding states on the ribosome.

Answer 30

As RNA binds independently to the subunits moving there can be variation in the sites the t-RNA binds to.

aa-t-RNA bind to A on the 30S and T in the 50S
Then normal conformation wherebind to A in both subunits so a peptide bond forms
hybrid form again of A/P and P/E due to spontaneous movement so half of aa-tRNA is on P site
EF-G mediates peptyltRNA out and aa-t-RNA over to P site.

Question 31

How important is GTP in ribosomal processing?

Answer 31

Need 1 GTP per initiation,

2 GTPs per elongation cycle,

1 GTP per termination event

GTP is an allosteric effector of the translation factors.

GTP hydrolysis expels the factors from the ribosome if correct (proofreading) Triggers GTP hydrolysis by EF-Tu and EF-G

Ribosomal GTPase centre
Involves 23S rRNA and proteins L7, L10, L11, L12

Inhibited by thiostrepton antibiotic.

Question 32

Why is elF2 important in EUKARYOTIC Initiation?

Answer 32

critical control point and is regulated by PHOSPHOYLATION

disassoiates with 40S subunit when 60S binds

Question 33

Why is IRES important in EUKARYOTIC Initiation?

Answer 33

IRES (internal ribosome entry site) allows initiation to happen through scanning mrna.

IRES bind upstream of AUG START SITE and begins scanning

Some virus can bind to 40S without any factors; virus cleave detected cap from mRNA so cell only makes viral proteins