Single-molecule studies of eukaryotic and prokaryotic transcription [electronic resource]

RNA, an essential, versatile macromolecule present in all cells, serves as the messenger for protein synthesis, in addition to participating in catalysis and gene regulation. Transcription is the process by which the enzyme RNA polymerase unwinds DNA and transmits its information to RNA. Many transcription factors interact with RNA polymerase to regulate its function. In my thesis I explore some of the molecular motors responsible for carrying out vital processes in the RNA cycle. In eukaryotic transcription, the process of how RNA polymerase initiates the synthesis of RNA is poorly understood. Using optical tweezers, I monitored the initiation of a 32-protein, 1.5 megadalton RNA polymerase II pre-initiation complex (PIC). The processes of open complex formation, transcription start-site scanning, and transition to a stable elongation complex were observed in real time. Scanning was found to be carried out by an associated motor, the transcription factor IIH (TFIIH) translocase, and entailed rapid opening by the TFIIH of an extended 85-bp bubble, followed by the synthesis of a transcript corresponding to almost the entire length of this extended bubble, followed by promoter escape and a transition to elongation. After RNA polymerase has successfully initiated and entered productive elongation to synthesize the transcript of interest, transcription must be terminated in order to recycle the polymerase for reuse, while providing a means to regulate RNA levels. Rho termination factor is an essential hexameric helicase responsible for terminating 20--50% of all mRNA synthesis in E. coli. I used single-molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho-utilization (rut) site of the λtR1 terminator. My results are consistent with Rho complexes adopting two states, one that binds 57 ± 2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ± 2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. I find that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation. The RNA synthesized must eventually be degraded to maintain homeostatis in a cell. Bacterial ribonucleases (RNases) are essential for gene expression, allowing rapid RNA turnover. I directly observed translocation of two cucial processive exoribonucleases from E. coli, RNase R and polnucleotide phosphorylase (PNPase). My results establish that RNase R is highly processive, and that a single molecule of this enzyme can unwind over 500 bp of structured substrate. I find that this motion is interrupted by pausing or stalling events that hinder degradation of the RNA in a sequence-dependent manner. Separately, I report that the number of nucleotides traversed by PNPase through structured RNA is highly dependent on the A+U content of the substrate, and that removal of the KH- and S1-binding domains reduces the processivity of PNPase without affecting velocity. I establish that PNPase is capable of translocating through tens of nucleotides of structured substrate, present evidence that it unwinds dsRNA in 6-7 nt steps, and find that RNase R shows sequence-dependent pausing and termination behavior. This result, taken together with previously reported structural and biochemical data, supports an asymmetric inchworm mechanism of motion.

Keywords: Applied Physics.
Advisor: Block Steven M.
Greenleaf William James advisor. Thesis advisor
Kornberg Roger D.