Abstract
RNA synthesis is an essential conduit of genetic information in all organisms. After transcribing an RNA, the bacterial RNA polymerase (RNAP) must be reused to make subsequent RNAs, but the steps that drive RNAP recycling are unclear. Adequate recycling of RNAPs may be important in allowing bacteria to rapidly reprogram gene transcription in response to stress or changes in cellular environment. However, the molecular players and pathways by which this reprogramming occurs in cells remains to be elucidated. In this work, we investigate the post-termination RNAP recycling mechanism by the RapA ATPase, a prokaryotic homolog of the eukaryotic Swi2/Snf2 chromatin remodeling complex, and explore its functional role in facilitating stress-induced transcriptional reprogramming. In Chapter II, we used multi-wavelength single-molecule fluorescence microscopy in vitro to observe the dynamics of individual molecules of fluorescently labeled RNAP and RapA as they colocalized with DNA during and after RNA synthesis. We provide direct evidence that RapA uses ATP hydrolysis to remove RNAP from DNA after RNA release and reveal essential features of the mechanism by which this removal occurs. Kinetic analysis and modeling were performed to elucidate the process through which RapA locates the PTC and the key mechanistic intermediates that bind and hydrolyze ATP. In Chapter III, we investigate the partitioning of cellular RNAPs in cells as a function of RapA during stress-induced transcriptional reprogramming using mathematical modeling. We extend this model to consider the phenomenon of transcriptional coupling between operons and speculate that this coupling may act as a functionally redundant mechanism to the activity of RapA under certain conditions, thereby revealing a novel rationale for the non-essentiality of RapA in vivo. In Chapter IV, we collaborate with other scientists to reveal the structural mechanism and conformational changes between RNAP and RapA which drive the dissociation of RNAP from DNA; these predictions are then tested using single-molecule fluorescence microscopy. Taken together, these studies fill in key missing pieces in our current understanding of the events that occur after RNA is released and that enable RNAP reuse, and suggest that RapA helps to maintain the balance between global RNAP recycling and local transcription re-initiation.