The ternary complex (TC) of Elongation Factor Tu (EF-Tu), GTP, and aminoacyl-tRNA (aa-tRNA) evolved to decode mRNA on the ribosome both rapidly and accurately. In E. coli, there are 43 aa-tRNAs which are used to decode 61 sense codons; each tRNA having a different sequence of nucleotides and one of 20 esterified amino acids. These diverse substrates bind with similar affinities to EF-Tu, however, EF-Tu specifically recognizes the esterified amino acid and tRNA body with >5000 fold range in affinities. To achieve uniform affinity, each tRNA has evolved to offset the affinity of the esterified amino acid.

The focus of this dissertation is to: (1) Identify how EF-Tu specifically binds each tRNA to offset the affinity of the esterified amino acid. (2) To identify the selective pressures that ensure uniform affinity of aa-tRNAs to EF-Tu. To accomplish these goals I used equilibrium binding, single turnover kinetics, and bioinformatics approaches.

To identify how EF-Tu specifically binds each tRNA, a mutagenesis approach was used to identify a three base pair region in the T-stem whose sequence is responsible for the observed tRNA specificity. Using a simple thermodynamic model we show it is possible to accurately predict the relative affinity of any tRNA based upon the sequence of these three base pairs in the T-stem. By applying this model to all bacterial tRNAs, we find that the affinity of each tRNA observed in E. coli is conserved across all tRNA isoacceptors, suggesting thermodynamic compensation is an ancient adaptation conserved across bacteria.

To understand the selective pressures that ensure uniform affinity of aa-tRNAs to EF-Tu, aa-tRNAs with tighter and weaker affinities than cognate were assayed in decoding. For a weaker binding TC, the ability to form the complex is impaired, however, once formed it decodes mRNA equivalently to cognate. For a tighter binding TC, selection by the ribosome is rapid, but the release from EF-Tu•GDP is retarded making it rate limiting for decoding. Thus, the uniform affinity of aa-tRNAs to EF-Tu evolved due to a need to bind tightly enough to form a stable TC, but weakly enough to release into the ribosome.