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. 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 encountered in nature and 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 two tRNAs for glutamic acid (tRNAglu) may allow this mutant tRNA to base pair with certain termination codons and thereby suppress translational termination. Assume that the wildtype tRNAglu has a U residue at the tRNA wobble position that allows this single tRNA to base pair with both Glu codons and answer the following questions:

A. What is the nucleotide sequence of the wildtype anticodon of tRNAglu ?

(write the sequence in 5’è3’ polarity)

     Wildtype anticodon sequence:

B. A single point mutation of the wildtype anticodon can convert it to one with suppressor activity. What is the mutant anticodon sequence?

(write the sequence in 5’è3’ polarity)

             Mutant anticodon sequence:

C. At which stop codons would the mutant tRNAglu 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 tRNAglu.

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

Question A)

The codon for Amino acid glutamic acid(Glu) is 'GAG' or 'GAA', read in 5' ->3' direction.

So the wild type anticodon in the tRNAGlu should have a sequence of 'CUC' or 'UUC' in the 5' ->3' direction.

Question B)

The normal stop codon have a sequence of 'UAA' , 'UAG' and 'UGA'. So the anticodons required for these are 'UUA' , 'CUA' and 'UCA' . (these are read in 5'->3' direction)

The anticodon 'CUC' , if it undergoes a point mutation, it can lead to formation of 'CUA' , which is an anticodon for the stop codon 'UAG'. Another possiblity is, if the anticodon 'UUC' undergoes point mutation, it can lead to the formation of 'UUA', which is the anticodon for 'UAA' . Thus the mutant anticodon sequence is 'CUA' and 'UUA'

Question C)

The anticodon which posseses the suppresor activity after mutation is 'CUC' and 'UUC'. Thus it will show an affinity toward the stop codon 'UAA' and 'UAG'.

Question D)

This kind of mutation ultimately will lead to improper translation of a polypertide, which might actually be the product of a structural gene or housekeeping gene. Thus proper experssion of the gene is always required and if not present the organism will not survive. The proper expression of the gene is only possible if there are two copies of gene which code for tRNAGlu. The proper expression will be there if both are wild type gene of if one of this is mutant supressor and the other is wild type, where the wild type will compensate for the functional loss caused by the mutant supressor type.

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