Talk abstracts
Talk on Friday 03:15-03:30pm submitted by Oshadhi Jayasinghe
NusG-dependent RNA polymerase pausing is a common component of riboswitch regulatory mechanisms
Oshadhi T. Jayasinghe (Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802), Alexander V. Yakhnin (NCI RNA Biology Laboratory, Center for Cancer Research, NCI, Frederick, MD 21702), Mikhail Kashlev (CI RNA Biology Laboratory, Center for Cancer Research, NCI, Frederick, MD 21702), Paul Babitzke (Department of Biochemistry and Molecular Biology, Center for RNA Molecular Biology, Pennsylvania State University, University Park, PA 16802)
Abstract:
Transcription is regulated during initiation, elongation, and termination. Transcription elongation is punctuated by RNA polymerase (RNAP) pausing. Pausing provides time for diverse regulatory events such as folding of regulatory signals in the nascent RNA, as well as binding of protein factors and ligands to the transcription elongation complex and the nascent RNA. The general transcription elongation factors NusA and NusG dramatically affect RNAP pausing in vitro and in vivo. We previously used nascent elongation transcript sequencing followed by RNase digestion (RNET-seq) to examine the genome-wide effects of NusA and NusG on pausing in Bacillus subtilis. Through a comprehensive analysis of RNET-seq data from WT and ΔnusG strains, we identified 1600 strong NusG-dependent pauses across the B. subtilis genome. Approximately 20% (~300) of these pause sites were localized to the 5’ leader regions, suggesting a regulatory role for these pauses. In this study, we focused on pauses that were associated with known transcription attenuation/riboswitch regulatory mechanisms. Pause sites that mapped to five known riboswitches were in strategic positions preceding the critical decision point between formation of alternative antiterminator or terminator structures that dictate the attenuation outcome. In vitro transcription assays confirmed the presence of NusG-dependent pauses at these positions, and that pausing increased termination in the 5' leaders in the presence of the cognate ligand. Our results also indicate that NusG-dependent pausing reduces the concentration of the ligand required for efficient termination in the 5’ leaders and/or provides additional time for proper riboswitch folding. Expression studies using integrated lacZ transcriptional fusions indicates that NusG-dependent pausing participates in each riboswitch mechanism in vivo. We propose that NusG-dependent pausing allows critical structural rearrangement of the nascent RNA, thereby allowing aptamer-ligand interactions, and hence enabling cells to sense a broad range of ligand concentration for fine tuning of riboswitch-mediated attenuation mechanisms.
Keywords: Riboswitches, RNA polymerase pausing, Post-transcriptional regulation