Single-molecule studies on transcriptional elongation in prokaryotes and eukaryotes
- Transcription, the process of copying genetic information stored in DNA into RNA, is fundamental to life. It is carried out by an extraordinary nano-machine called RNA polymerase (RNAP). Transcriptional elongation, during which RNAP moves along the DNA, adding one nucleotide at a time to the RNA transcript, is highly dynamic and regulated. The motion of RNAP is discontinuous and interrupted by pauses that play an essential role in gene regulation. Fundamental questions regarding the mechanisms of elongation and its modulation by transcription factors, however, remain unclear. In this dissertation, I focus on using high-resolution, optical trapping techniques to study the mechanisms of transcriptional elongation by both prokaryotic and eukaryotic RNA polymerases at the single-molecule level. First, I describe the studies on how the motion of single E.coli RNAP molecules is modulated by two universally conserved, essential transcription factors (NusA and NusG). From individual transcriptional elongation records, the rates of entering pause states, the pause state lifetimes, and the pause-free elongation speeds can all be extracted. By studying the effects of NusA (and NusG) on these kinetic rates as a function of the applied load, we were able to develop a quantitative kinetic scheme for elongation and pausing. This model not only explains the functions of NusA/NusG, but also provides insight into the mechanism of transcriptional pausing, which had previously been controversial. Second, a novel optical-trapping assay capable of directly probing elongation by individual eukaryotic RNA polymerase II (RNAPII) molecules will be described. We find that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, not only affects each of the three main phases of elongation, namely: substrate binding, translocation, and catalysis; but also plays a critical role in controlling the fidelity of transcription. Our data also support a Brownian ratchet model for elongation which incorporates a secondary nucleotide binding site.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Stanford University, Department of Applied Physics
|Kornberg, Roger D
|Kornberg, Roger D
|Statement of responsibility
|Submitted to the Department of Applied Physics.
|Thesis (Ph.D.)--Stanford University, 2012.
- © 2012 by Jing Zhou
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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