| Escherichia coli protein synthesis requires that 43 elongator aa-tRNAs quickly and accurately decode their corresponding 61 sense codons. Each aa-tRNA is chemically and structurally distinct from the other 42 aa-tRNAs due to its specific combination of sequence identity, length, esterified amino acid, and post-transcriptional modifications. While many mechanistic and structural details of mRNA decoding have been determined, most studies have been performed using only Phe-tRNAPhe. Therefore, the aim of this work was to compare the decoding properties of many aa-tRNAs and to determine if the structural elements of individual aa-tRNAs contribute to their decoding function.;To accomplish this goal, biochemical assays using [3'32P] labeled aa-tRNA were either created or adapted to facilitate the analysis of many aa-tRNAs. The kinetic and thermodynamic values of 10 different aa-tRNAs were then measured in steps of the decoding pathway. Despite the fact that each aa-tRNA was composed of a distinct set of structural elements, each aa-tRNA had similar affinities for the ribosomal entry site, rates of GTP hydrolysis, and rates of peptide bond formation.;To determine if the structural elements in an aa-tRNA tune it to perform uniformly with other aa-tRNAs on the ribosome, a highly conserved base pair in tRNAGGC Ala was substituted with base pairs commonly found in other tRNA sequences. The mutant Ala-tRNAGGC Ala (A32U-U38A) had similar decoding properties to the wild type Ala-tRNA GGC Ala on the cognate codon. However, unlike the wild type Ala-tRNAGGC Ala, Ala-tRNAGGC Ala (A32U-U38A) was also able to decode near-cognate codons corresponding to Valine and Threonine tRNAs.;The affect of the esterified amino acid on the decoding properties of an aa-tRNA was tested by misacylating tRNAGCC Gly with phenylalanine. On cognate codons, both correctly acylated Gly-tRNA GCC Gly and misacylated Phe-tRNAGCC Gly exhibit similar decoding properties. There were indications that Phe-tRNAGCC Gly has reduced decoding accuracy on near-cognate codons compared to Gly-tRNAGCC Gly, although the data was too variable to conclusively prove this.;These results have helped to demonstrate that the complete aa-tRNA, and not simply the anticodon, plays an essential role in accurately recognizing the mRNA codon to preserve the fidelity of protein synthesis. |
