Poster abstracts

Poster number 58 submitted by Rebecca Rohlfs

Mass spectrometry characterization of ribosome assembly defects

Rebecca L. Rohlfs (University of Cincinnati), Romel P. Dator (University of Cincinnati), Steven T. Gregory (Brown University), Al E. Dahlberg (Brown University), Patrick A. Limbach (University of Cincinnati)

Abstract:
The goal of this research is to examine ribosome assembly defects using mass spectrometry-based methods. The aim of this project is to use mass spectrometry to identify the post-transcriptional RNA modifications and post-translational ribosomal protein modifications that are unique or absent from improperly folded ribosome assembly subunits.
Escherichia coli is being used as the model system. Two strains of E. coli (K-12 and SK5665) are grown in the presence and absence of erythromycin at 25 ^oC and 37 ^oC. Subunits, intermediate, and intact 70S are separated and isolated by use of sucrose fractionation. Post-transcriptional modifications to RNA are identified using liquid chromatography with ultraviolet absorption (LC-UV) and mass spectrometry (LC-MS). Ribosomal proteins are identified, and any post-translational modifications confirmed, using matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS).
When erythromycin sensitive cells are cultured in the presence of erythromycin, the 50S subunit is unable to properly form, resulting in an intermediate 50S particle which sediments between 30S and 50S. The assembly particle is composed of both 5S and 23S rRNA and an undetermined number of large subunit ribosomal proteins. In wild-type cells, this mis-folded particle is degraded by the RNase E complex. E. coli strain SK5665 cells contain a temperature-sensitive RNase E phenotype, and RNase E can be inactivated by culturing at 25 ^oC, thus allowing this mis-folded 50S intermediate to be accumulated and isolated by sucrose fractionation. Currently, we have been characterizing the rRNAs and ribosomal proteins present in this mis-folded 50S intermediate and comparing their identity and modification status to the rRNAs and ribosomal proteins found in properly assembled 50S subunits. These data will provide insight into differences at the primary structure level between ribosomal subunits that can correctly fold with those that do not fold correctly.

Keywords: ribosome assembly, rRNA, liquid chromatography-mass spectrometry