Bisulfite-induced C changed to U transitions in yeast valine tRNA

Abstract

The reaction of yeast tRNAVallab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Six reactive residues have been located. C-17 in loop I is the most reactive (51% conversion) and C-73 in the first base pair of the acceptor stem the least reactive (8%). Three of the remaining reactive residues (C-39 in loop II, C-75 and C-76 near the acceptor stem) react to the same extent (36 to 38%) under the conditions of the experiment. C-37 in the anticodon reacted to a lesser extent (28%) than C-39 (36%), located just 2 residues away in the anticodon loop. No other changes were detected, but kinetic data suggest one or more additional residues may react very slowly. The C changed to U change in the anticodon (iac changed to iau) is a missense change (Val changed to Ile). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. We interpret the quantitative changes in tRNAVal to indicate that C-17 spends a large portion of its lifetime in an unstacked conformation. The stacking lifetimes of C-37, C-39, C-75, and C-76 seem to be similar but not identical. All other cytidine residues are much more tightly stacked. These results are consistent with the folded cloverleaf models that have been proposed from x-ray diffraction studies of yeast tRNAPhe. Residues C-46, C-49, C-57, and C-61, which are present in the single-stranded regions of the unfolded cloverleaf structure, do not react, suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also suggest that anticodon-loop conformations other than the extremes with five bases stacked on either the 3' or 5' portion of the anticodon stem exist in solution and that the anticodon loop is flexible.