Poster abstracts

Poster number 17 submitted by Romel Dator

Isolation and Characterization of Ribosome Assembly Intermediates

Romel Dator (Department of Chemistry, University of Cincinnati), Rebecca Rohlfs (Department of Chemistry, University of Cincinnati), Steven Gregory (Department of Biology, Brown University), Al Dahlberg (Department of Biology, Brown University), Patrick A. Limbach (Department of Chemistry, University of Cincinnati)

Abstract:
The long term goal of this research is to understand the significance of posttranscriptional modification in ribosomal RNA (rRNA) and posttranslational modification in ribosomal proteins (r-proteins) within ribosome assembly defects. In particular, mass spectrometry-based approaches will be used to characterize and identify the components of a transient ribonucleoprotein complex that is formed when bacterial cells are treated with the antibiotic erythromycin.

Binding of erythromycin to the ribosome can both inhibit protein translation and the assembly of the ribosome. Specifically, it interferes with the assembly of the 50S subunit leading to a stalled intermediate which is a target of degradation by cellular RNases. The use of the mutant SK5665 strain of E. coli which has a temperature-sensitive RNase E phenotype, allows for the isolation of the improperly folded assembly intermediate. Initial MALDI-MS data of the r-proteins isolated from the intermediate showed the absence of several proteins and was confirmed by SDS PAGE electrophoresis. Optimal growth conditions for growing the mutant SK5665 strain as well as the purification of the intermediate by sucrose density gradient ultracentrifugation will be presented. Further work is focused on determining whether the ribosomal proteins present in the improperly folded assembly intermediate differ from those found in properly assembled 50S subunits, both in identity and modification status.

Keywords: ribosome assembly intermediates, ribosomal proteins, mass spectrometry