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A sequence element that tunes Escherichia coli tRNAAlaGGC to ensure accurate decoding

Abstract

Mutating the rare A32-U38 nucleotide pair at the top of the anticodon loop of Escherichia coli tRNAAlaGGC to a more common U32-A38 pair results in a tRNA that performs almost normally on cognate codons but is unusually efficient in reading near-cognate codons. Pre–steady state kinetic measurements on E. coli ribosomes show that, unlike the wild-type tRNAAlaGGC, the misreading mutant tRNAAlaGGC shows rapid GTP hydrolysis and no detectable proofreading on near-cognate codons. Similarly, tRNAAlaGGC mutated to contain C32-G38, a pair that is found in some bacterial tRNAAlaGGC sequences, was able to decode only the cognate codons, whereas tRNAAlaGGC containing a more common C32-A38 pair was able to decode all cognate and near-cognate codons tested. We propose that many of the phylogenetically conserved sequence elements present in each tRNA have evolved to suppress translation of near-cognate codons.

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Figure 1: Secondary structure of E. coli tRNAAlaGGC.
Figure 2: Comparison of tRNAAlaGGC (wt) to tRNAAlaGGC (UA) on the GCC cognate and GCA near-cognate codons.
Figure 3: Time course of peptide bond formation for tRNAAlaGGC (wt) and tRNAAlaGGC (UA) on the cognate GCC codon (taken from Fig. 2d) and the mismatched ACC and GUC codons.

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Acknowledgements

We thank H. Suga and H. Murakami for discussions and for sharing their unpublished data. M.O. is supported by the “Homing” grant from the Foundation for Polish Science. This work was funded by the US National Institutes of Health grant GM037552 (to O.C.U.)

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S.L. and M.O. performed the experiments; S.L., M.O. and O.C.U. contributed to the design of the study and preparation of the manuscript.

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Correspondence to Mikołaj Olejniczak.

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Ledoux, S., Olejniczak, M. & Uhlenbeck, O. A sequence element that tunes Escherichia coli tRNAAlaGGC to ensure accurate decoding. Nat Struct Mol Biol 16, 359–364 (2009). https://doi.org/10.1038/nsmb.1581

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