Recent advances have brought the unexpected conclusion that transfer RNA specificity may have evolved before the addition of the anticodon domain, and thus predated this molecule's role in decoding mRNA. This suggests that tRNA may have originally had other primary function(s), and indeed the varied involvement of tRNA biology in the replication of RNA genomes, principally viral, has led to the suggestion that tRNA's original role was replicational in the RNA world. These conjectures heighten the importance of understanding the non-translational roles of tRNAs and tRNA-like structures (TLSs), in order to better understand RNA replication and the varied functions of tRNA, and to appreciate the possibilities for novel pathogenic effects (e.g. during persistent, sub-symptomatic infection by an RNA virus). The goal of this research is to obtain a detailed understanding of the role of a valine-specific TLS that is present at the 3' end of the turnip yellow mosaic virus (TYMV) RNA genome. This TLS has a novel structure in which a pseudoknot replaces the canonical antiparallel helix of tRNA to form the aminoacyl acceptor stem. It plays a role in genome replication that requires aminoacylation and does not involve priming (as with retroviruses), but whose details are not known. The investigator showed that the valine specificity can be replaced with methionine, and that the promoter directing (-) strand synthesis is present within the TLS. The proposed experiments address the role of tRNA mimicry with several complementary approaches. They will define the enzymological properties and the subunits of the RNA dependent RNA polymerase activity that is able to copy the TLS to produce (-) strands, identify proteins that bind to, or recognize elements in, the TLS and attempt to relate the results to the fact that the (-) strand promoter is a tRNA variant. They will attempt to reproduce the observed dependence of in vivo replication on aminoacylation in in vitro replication systems, so that the precise part of the replication cycle that depends on the ability of a template to be aminoacylated can be determined. A chimeric RNA, TYMC-ELV, that encodes wild type TYMV proteins and has a heterologous 3' end, will be investigated to determine why it replicates to high levels in plants although its RNA appears not to be aminoacylatable.
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