Studies on the Interactions of the Ribosome and RNA Polymerase and its Effect on Transcription in E coli
- Diggs, Stephen
- Advisor(s): Blaha, Gregor
RNA polymerase and the ribosome are two of the most well-studied macromolecules in all of molecular biology. Most research done on RNA polymerase and the ribosome has been done in isolation from each other. However, the cellular processes the RNA polymerase and ribosome carry out, transcription and translation, respectively, occur in the same cellular compartment in bacteria. In fact, the process of transcription and translation are coupled. Despite a detailed understanding of various aspects of transcription and translation, the coupling of both processes has remained enigmatic. This includes the interaction interface between the RNA polymerase and the ribosome, and between the RNA polymerase and the ribosomal subunits. Furthermore, we know how only a few ribosomal proteins influence transcription, a need for additional detailed studies are required to elucidate their potential effects on transcription during coupling. In my thesis, I first aimed to determine the interaction interface between RNA polymerase and the ribosome and between the RNA polymerase and the small and large ribosomal subunit. To do this, RNA polymerase was cross-linked with either the ribosome or one of the ribosomal subunits. The mixture was run on an SDS polyacrylamide gel, the crosslinked protein bands were excised from the gel, and the crosslinked proteins were identified in collaboration with Dr. Wang groups in the Chemistry department at UC Riverside. Based on this analysis we could identify several proteins on the ribosome that may participated in different interactions between the RNA polymerase and ribosome during transcription-translation coupling. For three of these ribosomal proteins, I determined their effects on transcription. Each ribosomal protein exerted a unique and unexpected effect on transcription. They all slowed the RNA polymerase at the his pause sequence. Two of them even hampered factor-mediated transcription termination, while one bound directly to core RNA polymerase. To enable a smooth follow-up of my initial studies of the ribosomal proteins' effect on transcription, I have included an extended appendix that details all aspects critical to performing these transcription assays successfully.