Synthesis of Tritium Labeled Queuine, PreQ1 and Related Azide Probes Toward Examining the Prevalence of Queuine

Queuine is a modified nucleotide known to occur in the anticodon of four tRNAs. The queuine modification occurs across all eukaryotes and eubacteria with few exceptions, but its function remains unclear. Prior in vitro work demonstrated that the modification can be incorporated into RNA species other than presently known tRNAs. Queuine is unusual in that, unlike the majority of modified nucleotides that result from changes to genetically encoded bases, it is incorporated into RNA by transglycosylation. Base modification by transglycosylation is unusual and represents an interesting point of entry for study. Due to this method of incorporation the modification can be studied with small molecule probes. Tritium-labeled queuine and preQ1 were prepared to study the differences between the eukaryotic and eubacterial version of the enzyme responsible for the incorporation of queuine, tRNA guanine transglycosylase. A concise, convergent synthesis of queuine was developed that is the shortest route to date.;PreQ1, the precursor to queuine incorporated by eubacteria, was used to investigate the prevalence of base modification in E. coli. Three cell lines were utilized to conduct the in vivo experiments of this study: a DeltaqueC knockout of E. coli that is unable to synthesize preQ1, so that labeled compound would be incorporated exclusively, a Deltatgt knockout strain of E. coli that is unable to incorporate preQ1 and a wild-type E. coli strain. As the modified nucleotide occurs through incorporation of a specially synthesized nucleotide, a study of the sites of modification by prepared probes is possible. The syntheses of two novel azide congeners were undertaken for this purpose. The evaluation of their interaction with tRNA guanine transglycosylase was undertaken to determine if they are substrates of the enzyme.;The tritium labeled preQ1 allowed for a general evaluation of the prevalence. That determined the four known tRNAs are the main site of incorporation in vivo, while many other RNAs are substrates in vitro. The azide probes were generated and interact with E. coli TGT, but were not substrates. In summary, we have gained a better understanding of queuine modification of RNAs and have developed tools that will aide in future studies.