Abstract
The reaction of yeast tRNAAla1ab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Five reactive residues have been located. Four of these (C-17 in Loop I, C-36 in the anticodon, C-74 and C-75 near the acceptor end) react to the same extent (42%) under the conditions of the experiment. The other (C-72 in the first base pair of the acceptor stem) reacts much more slowly (8%). No other changes were detected, but kinetic data suggest two or more additional residues may react very slowly. The C changed to U change in the anticodon (igc changed to igu) is a missense change (Ala changed to Thr). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. The quantitative changes for tRNAAla indicate that the stacking lifetimes of C-17, C-36, C-74, and C-75 are about equal. 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 48 and 56, which are in single-stranded regions in the unfolded cloverleaf structure, do not react suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also indicate that the anticodon loop is flexible in solution.
Highlights
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%)
37 in the anticodon reacted to a lesser extent (28%) than C-39 (36%), located just 2 residues away in the anticodon loop
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 tRNA”‘“
Summary
Yeast tRNAy:A was purified as described previously [12] and had a specific activity of 1,825 pm~l/A,,~,, unit, The material is a mixture of two isbacceptors differing by uridineidihydrouridine (59:41) modification at position 48 [12]. The assay was carried out as described previously [14] at the following final concentrations: Tris.HCl, pH 7.0, 60 mM; MgCl,, 30 mivr; EDTA, pH 7.0, 1.2 mivr; ATP, 5 mrvr; n-[‘%lvaline, 0.2 mM; tRNA, 6.5 x lo-” mM; purified enzyme, 6.5 milliunits/ml; final volume, 0.125 ml. Each reaction mixture was 100 ~1 with the following concentrations: tRNA, 2.5 x 10m5s (1.4A,,, units); MgCl,, 2.8 x. One portion of this solution (-0.1 A,,,, unit) was analyzed for its nucleoside composition as described previously [12]. At significantly lower enzyme concentrations, a considerable amount of the intermediate oligonucleotide, C-V-U-I>, was found This oligonucleotide does not separate well from the trinucleotide, D-C-G- The remaining chromatographic procedures and the quantitative measurements were carried out as described previously [1]
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