Recognition of tRNA analogues by the post-transcriptional hypermodification enzyme: tRNA-guanine transglycosylase.

In Escherichia coli, tRNA-guanine transglycosylase (TGT) is the enzyme which catalyzes the replacement of guanine-34 in the tRNA anticodon with preQ$\sb1$. Since tRNAs contain a large degree of conserved sequence and common three dimensional structure, discrimination between substrate and non-substrate tRNAs is a critical process. To investigate tRNA substrate recognition by TGT, several oligoribonucleotides have been generated using in vitro and in vivo systems. The tRNAs have been assayed with TGT to determine which portions of the tRNA are required for enzyme activity. The TGT reaction occurs among the final steps in tRNA maturation and tRNA recognition may require penultimately-modified tRNA. If other modified bases are recognized by TGT, then in vitro-generated tRNAs may exhibit reduced activity with TGT since they contain no modified bases. It was demonstrated that TGT could modify these in vitro-generated analogues and work was undertaken to determine specific kinetic parameters. Early data indicated that when almost 80% of the tRNA is removed, as in a 17 base anticodon loop minihelix, only a 10 fold decrease in activity from the full-length tRNA is found. Furthermore, D loop/anticodon loop and extended anticodon arm minihelices retain 30% of the original activity, indicating that only a fraction of the tRNA sequence is required for recognition by the enzyme. This also suggests that the structural motifs necessary for the interaction include a short helical region linked to a loop region. To gain further insight into the structural requirements an additional minihelix, with a three base pair stem extension of the anticodon arm of yeast tRNA$\sp{\rm Asp}$ was generated and characterized. Several sequence mutants of this analogue, focusing on the G30U40 base pair and U33G34U35 loop sequence, were produced and kinetic parameter determinations were performed. The results from kinetic evaluations of these analogues confirmed earlier homology studies which suggested that the U33G34U35 sequence is the major sequence determinant for TGT recognition. The overall conclusion of the results is that the major recognition elements in tRNA for TGT catalysis to occur are the U33G34U35 sequence in a loop region attached to a stable helix.PhDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104607/1/9542819.pdfDescription of 9542819.pdf : Restricted to UM users only