Grant: $75,000 - National Institutes of Health - Sep. 29, 2009
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Award Description: We have updated the Queuosine (Q) subsystem in the SEED database and analyzed 727 genomes for the presence of tgt, the signature gene of the Q pathway encoding tRNA guanine transglycosylase (TGT), and the precursor biosynthesis genes (queCDEF) . As shown in Fig 1A, approximately 25% of sequenced bacteria lack the precursor genes but contain a tgt gene. We predict that these organisms must rely solely on a salvage pathway in analogy to eukaryotes. The nature of the salvaged base or nucleobase, as well the transporters and enzymes involved in salvage, have yet to be identified and experimentally validated in any organism. The focus of this supplemental request is the elucidation and characterization of the transport and salvage pathways relevant for the biosynthesis of queuosine modified tRNA. More specifically, the following aims will be pursued Aim 1 The predicted Q transporter and nucleoside hydrolase genes will be deleted in several model organisms. Some, like Streptococcus pneumoniae, are predicted to rely exclusively on salvage. Others, like Escherichia coli ?queC, are mutated so the de novo pathway is no longer functional. The Q content in bulk tRNA will then be analyzed to test the role of predicted genes in Q salvage. In parallel, the expression of putative salvage genes will be measured by quantitative PCR in cells grown in the presence or in the absence of the queuosine precursors Aim 2 Genes that encode 2 putative nucleoside hydrolases implicated from comparative genomic analysis in queuosine salvage will be cloned and expressed and the corresponding recombinant enzymes characterized for their role in queuosine salvage.
Project Description: The focus of this supplemental request is the elucidation and characterization of the transport and salvage pathways relevant for the biosynthesis of queuosine modified tRNA. More specifically, the following aims will be pursued
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