Summary Transfer-RNAs (tRNA) are key molecules of translation, and their ability to accurately and efficiently decode genetic information is dependent on post-transcriptional modification of nucleotides, particularly in the critical anticodon stem loop (ASL). Deficiencies in these modifications can be lethal, and have been linked to a variety of pleiotropic phenotypes and human disease states. The long-term goals of this research program are to develop a detailed understanding of the biosynthetic pathways to complex tRNA modifications, the roles these modifications play in cellular physiology, and to identify novel targets in their pathways for therapeutic intervention. This application specifically focuses on elucidating the molecular mechanisms of formation and specificity of the universal ASL modification threonylcarbamoyladenosine (t6A) in bacteria, and elucidating the regulatory pathways that link it to bacterial cell wall synthesis. t6A is a complex modification found in the ASLs of tRNAs decoding ANN codons, and is critical for tRNA function by preventing ribosomal frameshifting, promoting cognate codon recognition, facilitating tRNA translocation, and serving as a recognition determinant for aminoacyl-tRNA synthetases. In the previous funding period we arrived at the first mechanistic proposal for the t6A biosynthesis cycle in which the proteins TsaC2, TsaB and TsaD function together to install threonylcarbamoyl on A37 of substrate tRNA, while TsaE provides an unexpected ATPase activity required for turnover of the cycle. We also discovered that TsaE is a novel bacterial S/T/Y kinase, and our bioinformatic analyses suggested a linkage of t6A biosynthesis to cell wall metabolism, which raises new questions about the widely reported cell wall synthesis phenotypes associated with t6A deficiency. In the current application we propose 4 specific aims that will allow us to elucidate 1) the tRNA specificity of the bacterial t6A system, 2) the mechanism of TC-AMP transfer in t6A biosynthesis, 3) the mechanism of ATP-hydrolysis driven turnover of the t6A cycle, and 4) the links between TsaE and cell-wall synthesis and/or cell division. This work will be accomplished through a combination of biochemical, structural, genetic, and physiologic approaches.