PhD (Doctor of Philosophy)
Date of Award
Committee Chair or Co-Chairs
W. Scott Champney
Phillip R. Musich, Lee M. Pike, Foster Levy, David A. Johnson
Erythromycin has long been recognized for its ability to inhibit protein synthesis by interfering with mRNA translation on the bacterial ribosome. We have recently shown that erythromycin also inhibits the assembly of the 50S ribosomal subunit in growing bacterial cells. The nature of this assembly inhibition has been investigated using 3H-uridine pulse-chase labeling of control and erythromycin treated E. coli cells.
Subunit assembly was examined by sucrose gradient centrifugation of labeled cell lysates. Normal assembly kinetics of subunit assembly were observed in control cells at 37°C. Formation of the 30S subunit was completed by 7.5 minutes and assembly of the 50S subunit was finished by 15 minutes after an unlabeled uridine chase. At 37°C, in the presence of erythromycin, 30S subunit assembly was unaffected but 50S assembly was greatly reduced. When the assembly kinetics were examined at 27°C, the assembly of both subunits was slowed and 30-32S precursor particle was seen to accumulate. This particle was found to bind 14C-erythromycin in vivo and in vitro.
RNase E has been implicated in the normal degradation and turnover of rRNA. A RNase E-mutant accumulated substantially more precursor to the 50S subunit than did control cells at either 37°C or 27°C. This precursor particle was also found to bind 14C-erythromycin. Specific 50S proteins and the 23S and 5SrRNAs were found in the 30S gradient region from lysates of cells grown at 27°C, confirming the presence of a 50S subunit precursor co-sedimenting with 30S subunits under these conditions. The precursor particle in the RNase E-mutant had a larger number of associated 50S proteins thandid the precursor from SK901. These data are consistent with our model of 50S subunit inhibition by erythromycin in which a fraction of 50S precursor particles undergo degraded.
Dissertation - Open Access
Usary, Jerry Edward, "Characterization of 50S Ribosomal Subunit Assembly Inhibition in Erythromycin Treated Escherichia coli Cells." (2000). Electronic Theses and Dissertations. Paper 20. http://dc.etsu.edu/etd/20
Copyright by the authors.