4. The termination of translation occurs when a ribosome that is translating an

Question

4. The termination of translation occurs when a ribosome that is translating an mRNA encounters a stop codon in the mRNA. Since there are no tRNAs that can base pair with stop codons, the A- site of a ribosome that is positioned at a stop codon is able to bind a protein (called release factor) that fosters hydrolysis of the bond linking the growing polypeptide to the tRNA currently occupying the ribosome’s P-site. But what if there was a tRNA that could base pair with a stop codon? Binding of this tRNA at the A-site could block release factor binding and - if the tRNA was “charged” with an amino acid - allow elongation of the growing polypeptide to continue past the stop codon. It turns out that particular point mutations in the anticodon loops of certain tRNAs can interfere with translational termination in just this way. Mutant tRNAs of this kind are referred to as suppressor tRNAs (for their ability to suppress translational termination). A particular single base substitution in the anticodon loop of one of the tRNAs for lysine (tRNAlys) may allow this mutant tRNA to base pair with certain termination codons and thereby suppress translational termination. Assume that the wildtype tRNAlys has a U residue at the tRNA wobble position that allows this single tRNA to base pair with both Lys codons that contain either a G or and A at the third nucleotide position of the codon and answer the following questions:

A) What is the nucleotide sequence of the wildtype anticodon of tRNAlys? (write the sequence in 5’3’ polarity)

Wildtype anticodon sequence:

B) A single point mutation of the wild type anticodon can convert it to one with suppressor activity. What is the mutant anticodon sequence?
(write the sequence in 5’3’ polarity)

C) At which stop codons would the mutant tRNAlys be able to suppress translational

termination? (remember that G-U base pairing is possible at the wobble position).

D) Explain why this particular mutation can only be harbored by bacteria that contain two

copies of the gene for tRNAlys.

E) Mutations in the anticodon loop do not always produce a functional suppressor tRNA due to the existence of a key step that contributes to translational fidelity that does not involve the ribosome. What is this step and how might it prevent suppressor tRNAs from being able to incorporate amino acids at stop codons?

F) Are there any other tRNAs for other amino acids that might be converted into suppressor tRNAs by point mutations in their anticodon loops? Give one example.

Explanation / Answer

A. The wildtype codon of Lys is UUU

B. The mutant anticodon with point mutation is UUA, which will bind to TAA stop codon.

C. The mutant anticon is able to bind to TAA, TAG stop codons.

D. Twp copies of this mutation will enable all the Lys codon to be encoded during the termination to enable the suppressor tRNA phenotype.

E. Faithful translation of mRNA into the corresponding polypeptide is a complex multistep process requiring accurate amino acid selction, transfer RNA charging and mRNA decoding on the ribosome. Key step in this process are aminoacyl-tRNA synthetases which not only catalyse the attachment of cognate amino acids to their respective tRNAs but also selectively hydrolyze incorrectly activated non-cognate amino acids or misaminoacylated tRNAs. This enzyme quality control provides proofreading that excludes non-cognate amino acids during translation and in so doing helps to prevent the formation of an aberrant proteome.

F. One of the example is Amber UAG suppressor Tyr mutation in which CUA anticodon is changed to GUA.

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