DOI
https://doi.org/10.25772/N4TX-W087
Defense Date
2010
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
H. Tonie Wright
Second Advisor
Jason P Rife
Abstract
Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the 30S ribosomal subunit, has been traced to the arm/rmt family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosylmethionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA in the 30S ribosomal subunit. Neither 16S rRNA alone nor intact 70S ribosome is an efficient substrate for armA methyltransferase. To more fully characterize this family of enzymes, xiii we have investigated the substrate requirements of ArmA. We determined the Mg2+ dependence of ArmA activity and found that the enzyme could recognize both translationally active and translationally inactive forms of 30S subunits. To identify the site of interaction between ArmA and the 30S subunit, we used hydroxyl radical cleavage of 16S rRNA mediated by ferrous iron chelated to several sites on the ArmA molecule that were mutated to cysteine. This data suggests that significant conformational changes in 30S structure are involved in binding of ArmA. We hypothesized that a precursor intermediate in the biogenesis of the 30S subunit might be the optimal substrate for ArmA enzymes in vivo. To test this, we prepared 30S particles partially depleted of proteins by treatment with increasing concentrations of LiCl and assayed them for ArmA methylation. Even low concentrations of LiCl alter the 30S particles and greatly diminish their susceptibility to methylation. Additionally, a previously identified pre-30S particle isolated from an E. coli culture was assayed for its ability to support methylation by ArmA and found to be inferior to intact 30S particles as a methylation substrate. Thus, testing of immature particles prepared from in vitro and in vivo sources suggest that ArmA works very late in the 30S biogenesis pathway. Initiation factor 3 (IF3), a factor that only binds fully mature 30S particles, does not inhibit the ArmA methylation, while kasugamycin methyltransferase (KsgA) abolishes ArmA activity by sharing the same binding site with ArmA. From aforementioned experiments, we conclude that ArmA is most active toward 30S ribosomal subunits that are at or very near full maturation.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
September 2010