Efficient mischarging of a viral tRNA-like structure and aminoacylation of a minihelix containing a pseudoknot: histidinylation of turnip yellow mosaic virus RNA

摘要

Mischarging of the valine specific tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA has been tested in the presence of purified arginyl-, aspartyl-, histidinyl-, and phenylalanyl-tRNA synthetases from bakers' yeast. Important mischarging of a 264 nucleotide-long transcript was found with histidinyl-tRNA synthetase which can acylate this fragment up to a level of 25% with a loss of specificity (expressed as V_max/K_M ratios) of only 100 fold as compared to a yeast tRNAHis transcript. Experiments on transcripts of various lengths indicate that the minimal valylatable fragment (n = 88) is the most efficient substrate for histidinyl-tRNA synthetase, with kinetic characteristics similar to those found for the control tRNAHis transcript. Mutations in the anticodon or adjacent to the 3' CCA that severely affect the valylation capacity of the 264 nucleotlde long TYMV fragment are without negative effect on its mischarging, and for some cases even improve its efficiency. A short fragment (n = 42) of the viral RNA containing the pseudoknot and corresponding to the amino acid accepting branch of the molecule is an efficient histidine acceptor.