EFFECTS OF THE RIBOSOMAL EXIT SITE ON TRANSLATIONAL ACCURACY

Abstract

Not all coding sequences are translated equally well, and this document describesstudies of sequence features that affect translation. Prior works suggest that codon:anticodon interaction in the ribosomal E-site helps maintain the reading frame. Here is tested the hypothesis that non-Watson-Crick codon:anticodon pairing at non- AUG initiation codons causes frameshifting. The results show that non-Watson:Crick pairing at the first codon position is not associated with frameshifting. The significance of the tRNA in translation is as an adapter molecule in delivering specific amino acids to matching codons on the mRNA chain. It has been suggested that correct discrimination of the correct aminoacyl-tRNA among approximately 60 competitors is accomplished in part by an allosteric interaction between tRNAs in the E-site and the A-site. This model suggests that the nature of the E site tRNA can affect the accuracy of aminoacyl-tRNA selection in the A site. Here is examined the effects, in vivo, of altering the E-site codon and assaying the effects on A-site misreading. Codons for essential amino acids codons in ß-Galactosidase were mutated such that enzyme activity requires misreading to insert the correct amino acid. Then, additional mutations at the E site position were made to determine whether the E site tRNA would affect misreading at the A site. It is shown that the E site codon can affect apparent misreading in some but not all tested sites. Moreover, it is shown that an AUG initiation codon in the E-site may dramatically increase A site misreading, leading to a hypothesis that sites that resemble translational initiation sequences may allow for high-frequency misreading of at least one codon (CAA misread as GAA). Finally, analyses of the entire E. coli genome sequence shows that codon and nucleotide biases differ for initiation and internal sequences, and strongly suggest that at least some of the initiation region bias has a translational cause. The described studies could provide valuable background for future work on sequence features that modulate genetic translation.