Lecture 2: The role of the ribosome in tRNA selection

Question 1

Why are the causes of mismatching of the codon-anticodon binding?

Answer 1

Charged tRNAs are selected by codon-anticodon interactions. They form a mini half-helix structure.
• Some correct interactions will only have 6 hydrogen bonds (e.g. UUU/AAA).
• Moreover, even in correct interactions, matching maybe imperfect because there are 61 separate tRNA species. These are wobble interactions.
• At third codon positions the G in the anticodon can match the U in the codon.
• Hypoxanathine in the anticodon can match A, C or U in the codon. In each case this only gives 2 hydrogen bonds.
• There can sometimes be very little difference in the binding energy of H bonds between correct and incorrect codon/anticodon interactions.

Question 2

What are the sources of miscoding?

Answer 2

There are multiple factors associated with miscoding, suggesting that different aspects of the structure are involved.
• Mutations in tRNA, especially at the elbow.
• Mutations in the ribosomal protein (S12, S5 and S4).
• Mutations in rRNA.
• Antibiotics.

Question 3

How do tRNAs undergo conformational change?

Answer 3

There are 3 conformations which tRNAs can undergo.

1) The ternary complex.
2) A*/T when the codon-anticodon binding first occurs. Occurs for cognate and non-cognate tRNAs.
3) A/T conformation. Adopted only by cognate tRNAs and requires a 30-degree bend in the tRNA body compared to the T conformation. Occurs in the A site. Bending is achieved by isolated distortion of the anticodon stem and the D stem. Mutations in the D stem can promote miscoding.

Question 4

What can mutations tell us about the mechanism of the ribosome in terms of speed, accuracy and important proteins?

Answer 4

The WT ribosomes have a compromise between speed and accuracy. Mutations can change this.
• Ram (ribosome ambiguity) are faster.
• Str (stringency or restrictive) are slower. They will reject some cognate aa-tRNAs and therefore waste GTP.
• S4, S5 and S12 are close to the decoding site and alterations to these proteins have an important effect on codon/anticodon interactions.

Question 5

What is the mechanism of the ribosome? How have we found it?

Answer 5

• There are 3 steps: the ribosome binds the ternary complex (independently of codon), codon is recognised and then the GTPase is activated.
• Translation fidelity results from the direct control of the GTPase by the coding centre.
• Cryo-EM and X-ray crystallography have given us a detailed mechanism of translation.
• Amino-acyl-tRNAs are delivered by the elongation factor EF-Tu. These are selected by ribosomes.
• In the initial binding is critical to distinguish cognate from non-cognate interactions, non-cognate interactions diffuse away.
• Both cognate and near-cognate tRNA anticodons explore the A site of an open 30S subunit.
• A transient conformation of G530 stabilises the cognate codon-anticodon helix.
• The 30S subunit closes. EF-Tu docks with the sarcin-rich loop (SRL) of the 50S subunit. This activates EF-Tu for GTP hydrolysis and enables accommodation of amino-acyl tRNA.
• Near cognates fail to induce the 530 latch, favouring 30S pre-accommodation intermediates with inactive EF-Tu. Inactive EF-Tu is separated from the SRL loop on the 50S subunit, preventing the activation of the GTPase.

Question 6

How are rRNAs involved in the ribosome?

Answer 6

• A1492 and A1493 position changes depending on the P and A sites.
• These residues form minor interactions with the codon-anticodon at the start site.
• They restrict the geometry of the first 2 nucleotides of the codon, forcing them into Watson-Crick like base pairs.
• 30S domain closure requires Watson-Crick base pairing at the first 2 codon-anticodon positions. G530 (semi-ON) forms H bonds with the codon-anticodon backbone.
• It then moves approximately 3 Angstroms (ON) and restructures the H bond network, forming H bonds with A1492 and positions 2 and 3.
• This leads to the shifting of the 30S shoulder. EF-Tu docks upon shoulder movement. This activates catalytic residue H84 to hydrolyse GTP.
• Upon GTP hydrolysis, the EF-Tu dissociates and cognate tRNA moves into the 50S A site.
• A near-cognate tRNA is inefficient in stabilising the G530 SEMI-ON state required to initiate 30S-domain closure. Equilibrium shifts to on conformation. This favours dissociation of the near-cognate ternary complex.

Question 7

What are the two ribosome conformations?

Answer 7

The ribosome has two conformations. The 30S subunit domains (particularly the shoulder) are involved. Only cognate tRNAs lead to 30S subunit closure.

1) Open conformation: E site is strong (high tRNA affinity) and the A site is weak.
2) Alternative conformation: A site is strong, and the E site is weak.