Project Summary Regulation of gene expression plays a key role in the cellular response to environmental damage by increasing the levels of specific stress response proteins. Recent tRNA modification, codon usage and proteomic studies provide support for the idea that stress promotes changes in the levels of enzyme-modified nucleosides found in the anticodon of some tRNA's to regulate the translation of stress-response transcripts with specific codon usage patterns. In bacteria the tRNA modification enzyme 2-selenouridine synthetase (SelU) has the potential to be exploited to develop new antibiotics, as SelU is essential in response to stress, unique to bacteria and adds a hydrophobic moiety, not found in humans, to the anticodon wobble uridine. In this application we propose to characterize stress-induced changes in SelU- dependent tRNA modifications and determine if regulation of translation elongation in bacteria is linked to tRNA modification and codon-usage patterns. Our studies in bacteria have the potential to demonstrate that specific tRNA modifications can promote and also restrict the translation of differing groups of codon-biased transcripts. Translational restriction would be a novel finding and could be linked to the observation that bacteria contain some unique RNA modifications when compared to eukaryotes. In contrast to eukaryotes, bacteria use 2-thiourdine (s2) as a substrate. Specifically SelU uses geranylphosphate to convert s2U to a geranylated wobble uridine (ges2U) in tRNAGlu, tRNAGln and tRNALys. SelU can also use selenophosphate to produce 2-selenouridine (se2U). Both ges2U and se2U are also found in the terminal modification 5- methylaminomethyl-2-geranylthioluridine (mnm5ges2U) and 5-methylaminomethyl-2-selenouridine (mnm5se2U), with mnm5 provided by another modification pathway. We have synthesized ges2U and se2U and used thermal denaturation and simulations to show that geranylation promotes wobble uridine pairing with guanine, while restricting pairing with adenine. We have used codon analytics of all E. coli and P. aerogenosis genes to identify codon-biased transcripts. Together with our observed stress sensitivity of E. coli ?selU cells, the data support the idea the ges2U and se2U modifications levels change in response to stress. Based on our studies we propose to test the hypothesis that the two SelU-based wobble uridine modifications in bacterial tRNA are dynamically regulated in response to environmental stress and can be used to positively- or negatively- regulate the translation of specific transcripts corresponding to stress response and metabolic enzymes, respectively. There is abundant s2U modified tRNA in human cells but its analysis is understudied, due to the absence of essential tools. Because SelU and ges2U are unique to bacteria and geranylphosphate is a 10-carbon chain, they have the potential to be exploited to label specific s2U-modified tRNAs in human cells. Thus we will test the hypothesis that SelU can be exploited to generate an s2U-based labeling reagent for use in epitranscriptomic studies of human cells.