Characterization of the methionyl-trna synthetase from mycoplasma penetrans

Resumen

The focus of this thesis is to characterize the aminoacylation activity of the methionyl-tRNA synthetase from Mycoplasma penetrans (MpMRS), determine the effect of its appended N-terminal domains on this activity, and determine differences in tRNA recognition and discrimination between the methionyl-tRNA synthetase of Escherichia coli (EcMRS) and MpMRS. An appended domain of MpMRS appears to be related to aminotransferase V domains and contains conserved residues for pyridoxal 5'-phosphate (PLP) binding. RT-PCR results indicate that the full-length transcript is present in vivo. However, Western Blot analysis using antibodies against synthetic peptide provides evidence that variants of the protein both with and without the appended domain are present in the cell. MpMRS and a Mp¿MRS mutant lacking the appended domains showed similar patterns of aminoacylation activity. Both enzymes methionylated M. penetrans tRNAMetCAU and tRNAfMetCAU transcripts, but, unlike EcMRS, they could not methionylate M. penetrans tRNAlleCAU transcripts. Efforts were made to identify the tRNA residues important for the novel tRNAlleCAU discrimination by MpMRS by evaluating the effects of tRNAlleCAU and tRNAMetCAU mutants on kinetic parameters of aminoacylation. A series of mutant tRNAlleCAU were synthesized with residues in the D-loop, T-loop, anticodon stem, and acceptor stem changed to the corresponding bases of tRNAMetCAU. Replacement of acceptor stem residues of tRNAlleCAu with those of tRNAMetCAu, especially the tRNAlleCAU (A3C-U70G) mutation, allowed the altered tRNA to be aminoacylated by MpMRS. Likewise, a converse set of acceptor stem mutations of tRNAMetCAU caused a decrease in methionylation of the tRNA by MpMRS. Microhelix experiments showed no fundamental differences between MpMRS and EcMRS in relation to microhelix aminoacylation or microhelix aminoacylation inhibition. Broadening the study of tRNAlleCAU discrimination to include MRS from Helicobacter pylori (HpMRS) and Streptococcus pneumoniae (SpMRS) revealed that HpMRS may discriminate against tRNAlleCAU, while SpMRS does not discriminate against S. pneumoniae tRNAlleCAU transcript. Finally, analysis of these sequences and MRS aminoacylation results suggest that further study is needed regarding the role of the zinc binding domain of MRS in tRNAlleCAU discrimination.