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Hon-Chiu Leung

Advisor: Malcolm Winkler, Ph.D.

The hypermodified, hydrophobic 2-methylthio-N6-(dimethylallyl)-adenosine (ms2i6A) residue occurs 3′ to the indication in tRNA species that read colons beginning with U. The first Step (i6A37 formation) of this modification is catalyzed by dimethylallyl diphosphate:tRNA dimethyallyl transferase (EC, which is the product of the miaA gene. Subsequent steps were proposed to be catalyzed by MiaB and MiaC enzymes to complete the ms2i6A37 modification. The study of functions of ms2i6A37 is very important because this modified base is one of the best candidates for a role in global control in response to environmental stress. This dissertation describes the further delineation of functions of the ms2i6A37 modification in E. coli K-12 cells. This work provides significant information on functions of tRNA modifications in E. coli cells to adapt to stressful environmental conditions. Three hypotheses were tested in this work.

The first hypothesis tested was that non-optimal translation processes cause increased spontaneous mutagenesis by the induction of SOS response in starving cells. To test this hypothesis, I measured spontaneous mutation rates of wild type cells and various mutant strains which are defective in TRNA modification, SOS response, or oxidative damage repair. These strains were grown under starving conditions, starving conditions together with sublethal concentrations of kanamycin, or exponentially growing conditions. The miaA mutation acts as a mutator that increased Lac+ reversion rates and Trp+ reversion frequencies of wild-type cells in starving conditions. However, the lexA3(Ind-)(which abolishes the induction of SOS response) mutation abolished the mutator phenotype of the miaA mutant. The recA430 mutation, not other identified SOS genes, decreased the Lac+ reversion to a less extent than that of lexA3(Ind-) mutation. These results suggest that RecA together with another unidentified SOS gene product are responsible for the process. Starving conditions together with sublethal concentration of kanamycin increased Lac+ reversion rates by the induction of SOS response. Therefore, these results proved the hypothesis that non-optimal translation processes can be a cause of spontaneous mutagenesis by the induction of SOS response in starving cells. However, the miaA mutation acts as an antimutator in cells growing exponentially in minimal glucose medium. The lexA3(Ind-) mutation counteracted the antimutator effect of miaA mutation.

The second hypothesis tested was that MiaA protein binds to full-length tRNA Phe molecules in form of a protein dimer. To test this hypothesis, three versions of the MiaA protein and seven species of tRNA substrates were purified. Binding studies by gel mobility shift assays, filter binding assays and gel filtration shift assays support the hypothesis that MiaA protein binds to full-length tRNA Phe a These results were further supported by using steady state enzyme kinetic studies.

The third hypothesis tested in this work was that the miaB gene in E. coli exists and is clonable. The miaB::Tn10dCm insertion mutation of Salmonella typhimurium was transduced to E. coli K-12 cells by using P1 and P22 bacteriophages. The insertion was confirmed by HPLC analyses of nucleotide profiles of miaB mutants of E. coli. >The insertion mutation was cloned and DNA sequences adjacent to the transposon were sequenced. These DNA sequences were 86% identical to the f474 gene at 14.97 min chromosome of E. coli. The f474 gene was then cloned by PCR from the wild-type chromosome of E. coli. The recombinant plasmid complemented the mutant phenotype of the miaB mutant of E. coli. In summary, functions of thems2i6A37 modification in E. coli cells are further delineated in this work in perspectives of adaptation to stressful environmental conditions and protein:tRNA interaction.

Search pubmed for papers by H Leung and ME Winkler

Research Info

2-Methylthio-N6-Dimethylallyl Modification of Adenosine 37 of tRNAs in Escherichia coli K-12