The molecular basis for high fidelity tRNA selection on the ribosome
Faithful translation of a messenger RNA (mRNA) into a polypeptide chain depends largely on the specificity with which the cognate aminoacyl tRNA (aa-tRNA) is selected during each round of protein chain elongation on the ribosome. The mechanism by which high-fidelity tRNA selection is achieved is complex, as it needs to account for high specificity as well as for high speed. Previous work has shown that an induced fit mechanism provides the required discrimination power. This mechanism involves a series of conformational changes induced only by the cognate aa-tRNA that result in selective acceleration of critical steps during the decoding process for the cognate but not for an incorrect aa-tRNA. The nature of the required conformational changes is not yet fully understood. By kinetically characterizing a variant tRNATrp we identified the body of the tRNA itself as a critical player in this selective rate acceleration, and thus as a critical component of the induced fit mechanism. We have also studied the contribution of conserved residues in the decoding site of the ribosome by carrying out a mutational analysis. Our results show that these nucleotides are essential for cognate aa-tRNA selection. In addition, we carried out a biochemical epistasis analysis combining these variant ribosomes with the mutant tRNATrp and antibiotics that are known to promote miscoding. These studies revealed at least two requirements for tRNA selection on the ribosome, which further experiments will help to define. Together, the results presented in this thesis increase our understanding of the molecular basis for high-fidelity decoding during protein synthesis on the ribosome.