Lecture 8. The Architecture of Translation

Question 1

What is translation?

Answer 1

Converts the genetic code (DNA) into protein sequence
The joining of aminoacyl residues by the ribosome to form a polypeptide
High energy cost to cell
Cells posses > 150 proteins and ~40 tRNAS which function in protein synthesis

Question 2

What are the similarities between prokaryotic and eukaryotic ribosomes?

Answer 2

Large RNA in both subunits in both types of ribosomes
Large and small subunits in both types of ribosomes
Large number of proteins associated with the RNA of the large and small subunit in each case

Question 3

What are the differences between prokaryotic and eukaryotic ribosomes?

Answer 3

Things are slightly bigger in the eukaryotic ribosome (80S vs 70S) and eukaryote ribosomes have more proteins
Mechanism is similar between the two

Question 4

What is the secondary structure of bacterial 16S rRNA?

Answer 4

Conserved regions of rRNA where mutations in these regions are fatal
Variable regions that can tolerate mutations
Base-paired stems (A-form helix) common - compensating base changes between species
Results in the same overall structure for all 16S rRNA

Question 5

How is the structure of 16S rRNA conserved?

Answer 5

Compensating base changes
A single point mutation that disrupted base pairing seen causes a second mutation that restores base pairing compensates

Question 6

What are the three binding sites for tRNA in the ribosomes that span the 30S and 50S subunits?

Answer 6

A = Acceptor site of codon-directed binding of incoming aa tRNA
P = Peptidyl site; holds codon directed peptidyl tRNA (formation of new peptide)
E = Exit site; not associated with mRNA (releases to pick up another tRNA and start process again)

Question 7

What happens in the 23S rRNA peptidyl transferase reaction?

Answer 7

Main point: Reaction in the ribosome is that of forming a new peptide bond
1. N3 of A2486 accepts a proton from the amino group of the aminoacyl tRNA in the A site
2. This enhances the negative charge of the amino group, allowing it to attack the bond between the peptide and tRNA in the P site
3. The N3 of A2486 H-bonds to the oxyanion in the tetrahedral intermediate stabilising it and accelerating the reaction. The 3’-OH of the tRNA in the P site accepts the proton from A2486, completing the reaction (ends with tRNA-OH)

Question 8

What is one of the most important things to remember about the 23S rRNA peptidyl transferase reaction?

Answer 8

The RNA is involved in tis reaction - riboszyme activity
Not protein, it is the RNA that is involved in the reaction

Question 9

What is the poly-peptide exit tunnel in the 50S subunit?

Answer 9

Tunnel between the peptidyl transferase active site and the exit site
Allows the peptide to come out as it is being formed, slippy which allows an α-helix to form within the exit tunnel
The protein being made is also important as keeps the two subunits of the ribosome together

Question 10

How are tRNAs (transfer RNAs) named?

Answer 10

tRNAs are named according to the amino acid with which they become charged - (Esterified) g.g tRNAala; following charging alanyl tRNA

Question 11

What are isoaccepting tRNAs?

Answer 11

Several different tRNAs (often with different anticodon sequences) that become charged with the same amino acid - common

Question 12

How is tRNA aminoacylated?

Answer 12

The aminoacyl-tRNA synthetases (The enzymes which charge tRNAs) show specificity for the tRNAs they charge, and the correct interaction is with cognate tRNAs
Very rarely, a non-cognate (“incorrect”) tRNA is aminoacylated

Question 13

What do tRNAs contain?

Answer 13

A number of modified (unusual) nucleosides
tRNAs have to be nearly identical to fit into pocket, but be able to attach to different areas
Dihydrouridine (DHU), Ribothymidine (T), Pseudouridine (Ψ), Inosine (I), ²N-Methylguanosine (mG)

Question 14

What is the cloverleaf model for tRNA?

Answer 14

Three main loops and a smaller loop
D loop on the left
Anticodon loop at the bottom
T (TψC) loop on the right
Smaller variable loop between anitcodon and T loop
Attached amino acid with at top with 3’ end always ending with CCA (A allows for amino acid attachment)
Stems between the loops

Question 15

What is the D loop on the cloverleaf model of tRNA?

Answer 15

D loop contains 8-12 unpaired bases -contains 2-3 dihydrouricil residues

Question 16

What is the anticodon loop on the cloverleaf model of tRNA?

Answer 16

Anticodon loop of 7 unpaired bases
Contains the three anticodon bases
The anticodon is flanked on its 5’ side by U, and on 3’ side by an alkylated purine

Question 17

What is the variable loop on the cloverleaf model of tRNA?

Answer 17

Varies in size

Question 18

What is the T (TψC) loop on the cloverleaf model of tRNA?

Answer 18

The TψC loop
7 unpaired bases
5’ TψCG 3’ present
Involved in binding to the ribosome ‘A’ site

Question 19

What is the attached amino acid in the cloverleaf model?

Answer 19

At the top
3’ end always has CCA
A allows for the attachment of an amino acid
4th base variable

Question 20

What are the stems between the loops on the cloverleaf model of tRNA?

Answer 20

Gives structure
Closely controls sizes

Question 21

What is the tertiary structure of yeast tRNA(Phe)?

Answer 21

CCA-3’ is located ~70 Å away from the anticodon
The DHU and TψC loops form the corner of the “L” 20 Å
Most bases are stacked, a major factor in stabilisation
The 3 anticodon bases and the -CCA-3’ bases are unstacked, allowing interaction with the codon base, or the aminoacyl-tRNA synthestase
Many of the tertiary H-bonding interactions involve bases that are invariant in all known tRNAS, strongly supporting the belief that all tRNAS have basically the same structure - many of these H-bonds involve non-conventional A-U and G-C base pairs

Question 22

What are the shared reactions of all tRNAs?

Answer 22

1. Interaction with elongation factor (except initiator tRNA) - elongation factor involved with translation
2. Binding to the ribosome ‘A’ site
3. CCA terminal addition
4. ‘Invariant’ modifications to base

Question 23

What are the unique reactions of individual tRNAs?

Answer 23

1. Amino acylation by synthetases
2. Codon-Anticodon interaction
3. Recognition of initiator (fmet tRNA) by initiation factor
4. Recognition of initiator by transformylase
5. Unique base modification

Question 24

How is tRNA charged by aminoacyl-tRNA synthetases?

Answer 24

1. A specific amino acid and ATP bind to the aminoacyl-tRNA synthetase
2. The amino acid is activated by the covalent binding of AMP, and phosphate is released
3. The correct tRNA binds to the synthetase. The amino acid is covalently attached to the tRNA. AMP is released
4. The charged tRNA is released

Question 25

What bond is involved with aminoacyl-tRNA linkage?

Answer 25

Ester bond between the amino acid and tRNA form

Question 26

What are cognate tRNAs?

Answer 26

The tRNAs that each charge the specific single amino acid recognised by a specific synthetase

Question 27

What may cognate tRNAs differ by?

Answer 27

May differ in their anticodons and in other parts of the molecule

Question 28

How diverse are aminoacyl-tRNA synthetase enzymes?

Answer 28

Range from 40 to 100 kDa in size
May be monomeric, dimeric or tetrameric

Question 29

What are the two general groups of aminoacyl-tRNA synthetases?

Answer 29

Class I and class II enzymes

Question 30

What is the difference between class I and class I synthetases in terms of their RNA contacts?

Answer 30

Class I Contacts tRNA at minor groove of the acceptor stem and anticodon
Class II Contacts tRNA at major groove of the acceptor stem and anticodon

Question 31

What is the difference between class I and class I synthetases in terms of their catalytic sites?

Answer 31

Class I catalytic site found at terminal and is made up of repeating α-β-α-β-
Class II catalytic site found in the central β-sheet surrounded by massive α-helix

Question 32

How does the CCA arm of the tRNA conform to class I and class II synthetases?

Answer 32

Because class I and II synthetases recognise different faces of the tRNA molecule, the CCA arm adopts different conformations with the two classes

Question 33

How important is it that the tRNA is charged by the aminoacyl-tRNA synthetases?

Answer 33

This is of equal importance to the accuracy of protein synthesis as the selection of the amino acid
The features of individual tRNAs which are recognised by their cognate synthetase are called identity elements
Logic would suggest that identity elements of the tRNA lie in its anticodon
bases. This is true for some tRNAs. However, for others, the identity elements lie
elsewhere
The significance of this is apparent from crystal structures of tRNA synthetase complexes

Question 34

Where does the tRNA synthetase interact with in yeast tRNA?

Answer 34

Interacts with base pair G10:C25 in addition to the acceptor stem and anticodon loop

Question 35

What happens when an amino acid binds to the CCA chain?

Answer 35

You get movement of the CCA chain, it is flexible and flex between the activation and editing site
If the amino acid fits between into the editing site than the activation site, the amino acid is cleaved off by hydrolysis because it is the incorrect amino acid
Zinc ion helps stabilise interactions

Question 35

How does proofreading by aminoacyl-tRNA synthetases occur?

Answer 35

Proofreading can occur at two stages (so-called double sieve)
1. By hydrolysis of the ester bond of an “incorrect” aminoacyl-AMP intermediate triggered by the binding of the cognate tRNA
2. By hydrolysis of the ester bond of a “miss-matched” aminoacyl-tRNA

Question 36

What do most aminoacyl-tRNA synthetases possess a that is required for proofreading?

Answer 36

Most aminoacyl-tRNA synthetases possess editing (hydrolytic) sites in addition to the acylation site
Usually, the acylation site rejects an amino acid larger than the cognate aa, due to insufficient room. The editing site hydrolyses aminoacyl-tRNAs which are smaller than the cognate aa
Overall fidelity of charging in vivo 99.99%

Question 37

What is an example of translation being targeted by antibiotics?

Answer 37

Several antibiotics that inhibit the peptidyl transferase activity of ribosomes (e.g. erythromycin and chloramphenicol) bind directly to the peptidyl transferase centre of the 23S rRNA. Single base mutations in the antibiotic binding sites results in antibiotic resistance

Question 38

What is streptomycin and how does it function?

Answer 38

Streptomycin is a highly basic trisaccharide
Streptomycin binds to the 16S rRNA of the 30S subunit of the bacterial ribosome, which interferes with the binding of formylmethionyl-tRNA to ribosomes and thereby prevents the correct initiation of protein synthesis

Question 39

What is puromycin and how does it function?

Answer 39

Puromycin resembles the amino acyl part of aminoacyl tRNA
Puromycin enters the vacant A site without the involvement of EF-Tu
It is a substrate for peptidyl transferase through its amino group forming peptidyl puromycin
Peptidyl puromycin is not anchored to the A site, and dissociates from the ribosome, resulting in premature chain termination
About 50% of ribosomes react with puromycin – those with vacant A sites

Question 40

What is diptheria toxin and how does it function?

Answer 40

Diphtheria toxin is produced by pathogenic strains of Corynebacterium diphtheriae. It is highly toxic with a mouse LD50 ~100 ng/kg
It acts catalytically on elongation factor 2 (EF-2), the eukaryotic homologue of EF-G
All EF-2s contain a postranslationally modified histidine residue called diphthamide. The toxin transfers ADP ribose from NAD⁺ to the imidazole ring. This completely inhibits translocation.

Question 41

How can ricin be used in threapy?

Answer 41

Combined targeting ability of an antibody (modified from PDB entry 1igt) with the toxicity of the ricin A chain (shown in red)
Will only kill cancer cells