Evolutionary tuning impacts the design of bacterial tRNAs for the incorporation of unnatural amino acids by ribosomes

Abstract

In order to function on the ribosome with uniform rate and adequate accuracy, each bacterial tRNA has evolved to have a characteristic sequence and set of modifications that compensate for the differing physical properties of its esterified amino acid and its codon-anticodon interaction. The sequence of the T-stem of each tRNA compensates for the differential effect of the esterified amino acid on the binding and release of EF-Tu during decoding. The sequence and modifications in the anticodon loop and core of tRNA impact the codon-anticodon strength and the ability of the tRNA to bend during codon recognition. These discoveries impact the design of tRNAs for the efficient and accurate incorporation of unnatural amino acids into proteins using bacterial translation systems.

Figure 1.. Steps in the mechanism of bacterial translational decoding

For a full description of the decoding mechanism and the values for the rate constants of individual steps, see Rodnina et al. [48]. High resolution x-ray crystal structures are available for each of the ribosome bound intermediates [39,50,51] except for the labile initial binding complex. Structural diagrams were generated using VMD [52].

Figure 2.. Structural elements in aa-tRNAs that contribute to EF-Tu binding specificity

A. Variable contributions of amino acid and tRNA body to EF-Tu affinity. ΔΔG° values for substituting either the amino acid (left) or the tRNA body (right) of Phe-tRNA^Phe are listed. Both substitutions are ranked from the most stabilizing (blue) to the most destabilizing (red), but are presented in inverse order to emphasize that the values compensate for correctly acylated tRNAs [6]. By using a ΔG°(total) = −10.1 kcal/mol for Phe-tRNA^Phe, the ΔG°(total) of any misacylated aa-tRNA can be calculated from these data [5]. Values are only appropriate for the experimental conditions used in [5] since EF-Tu binding affinity is very dependent on temperature and ionic strength. B. T-stem sequence affects aa-tRNA binding to EF-Tu. ΔΔG° values for substituting the C49-G65 (green), U50-A64(blue) and U51-G63 (red) pairs present in E. coli tRNA^Phe for other base pairs. Since these ΔΔG° values contribute independently from one another, these data can be used to calculate the ΔG°(tRNA) for any tRNA sequence [13] and combined with data in panel A to estimate ΔG°(total) for any aa-tRNA.

Figure 3.. Idiosyncratic evolutionary tuning of two E coli tRNAs.

Two tRNAs with quite different decoding strategies are compared. tRNA^Ala_GGC possesses an amino acid that binds EF-Tu weakly and has a very stable codon-anticodon interaction. tRNA^Tyr_GUA possesses a tighter binding amino acid and a weaker codon-anticodon interaction. While EF-Tu binding data for both tRNAs is well established, systematic mutagenesis of the anticodon stem and core has only been performed for tRNA^Ala_GGC [40]. Since no direct measurement of the flexibility of these tRNAs has been performed, these descriptions are speculative. Modified nucleotides are in bold [42], the EF-Tu recognition region is boxed in green and the anticodon is circled in blue.