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

Abstract

The conserved A32-U38 pair in the anticodon loop of tRNAAlaGGC is now shown to be important for accurate decoding. Pre–steady state kinetic analyses of mutants in A32-U38 show that they can efficiently decode near-cognate codons, with a mismatch in any of the three positions, pointing to the role of such conserved sequence elements in suppressing misreading during translation. 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.