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

Abstract

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 Vmax/KM ratios) of only 100 fold as compared to a yeast tRNA(His) 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 tRNA(His) transcript. Mutations in the anticodon or adjacent to the 3' CCA that severely affect the valylation capacity of the 264 nucleotide 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.

Identity elements ofSaccharomyces cerevisiaetRNAHis

“…These results raise the possibility that A73 has no direct interaction with histidyl-tRNA synthetase, and that other bases, particularly G73, behave as obstacles to the normal positioning of the CCA end in aminoacylation. This is in an apparent contradiction to another study showing that an A to G mutation at the putative discriminator position of a tRNA-like structure corresponding to the 3' end of the turnip yellow mosaic virus RNA does not seriously affect aminoacylation with yeast histidyl-tRNA synthetase (36). Particular impairment by G73 has also been observed in E.coli (7).…”

An operational RNA code for amino acids and possible relationship to genetic code.

“…1).] An RNA pseudoknot structure based on the 3' end of turnip yellow mosaic virus has also been enzymatically aminoacylated (28).…”

“…The numbering system is based on the tRNA molecule shown in A. The pseudoknot minihelix is based on an acceptor-TlC minihelix-like domain in turnip yellow mosaic virus (28).…”

Histidyl-tRNA Synthetase

“…Sequence similarities occur in the acceptor stem and the D loop region. The RNA of the turnip yellow mosaic virus (TYMV) is aminoacylated with valine by ValRS from yeast as well as with histidine by HisRS, albeit with lower efficiency (Rudinger et al, 1992). This indicates that the TYMV RNA contains essential identity elements recognized by both aaRS enzymes (for compilations see also Mans et al, 1991;Giegé, 1996).…”