Characterization of tRNA-guanine transglycosylase from Escherichia coli.

This thesis describes the characterization of tRNA-guanine transglycosylase (TGT, E.C.2.4.2.29) from Escherichia coli. TGT is involved in the posttranscriptional modification of tRNA. Our initial studies indicated that native TGT was trimeric in solution and it was dissociated into tRNA-bound monomer after incubation with the substrate tRNA. Chemical modification studies suggested that cysteine residues might be involved in TGT activity. TGT contains approximately stoichiometric zinc. Cysteines and histidines were proposed to serve as the zinc ligands. In order to further characterize TGT using the site-specific mutagenesis approach, an optimized protein expression vector (pTGTS) and an efficient site-specific mutagenesis method (the "preferential PCR" method) have been developed. All TGT mutants were overexpressed in the E. coli K12 ($\Delta{tgt})$ strain. All eight cysteines (C28, C145, C232, C265, C302, C304, C307 and C321) and two of the histidines (H316 and H317) in TGT have been mutated into alanine. Gross structural characterization of these mutants indicates that cysteine 302, 304, 307 and histidine 317 are the zinc ligands. These zinc ligand residues are conserved in the tgt genes from E. coli and Z. mobilis. The zinc-binding site in TGT appears to be important for maintaining the tertiary structure and enzymic activity. DTNB modification studies of TGT (C265S) mutant indicate that the inactivation of the wild-type enzyme by DTNB was caused by the modification of cysteine 265, and that cysteine 265 is located in or near the active site. Kinetic characterizations of both TGT (C265A) and TGT (C265S) suggest that cysteine 265 does not play a critical role in the catalysis. Kinetic characterizations of TGT (S90A) and TGT (S90C) mutants indicate that serine 90 plays a critical role in TGT catalysis. A serine-protease type mechanism is suggested for the TGT reaction. More experimental data, especially from X-ray crystallography studies, are needed in order to fully understand the molecular mechanism of the TGT reaction.Ph.D.PharmacognosyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104446/1/9527600.pdfDescription of 9527600.pdf : Restricted to UM users only