Single-molecule fluorescence analysis of opening and closing of the RNA polymerase clamp
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Chakraborty, Anirban.
Single-molecule fluorescence analysis of opening and closing of the RNA polymerase clamp. Retrieved from
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TitleSingle-molecule fluorescence analysis of opening and closing of the RNA polymerase clamp
Date Created2013
Other Date2013-01 (degree)
Extentxiii, 111 p. : ill.
DescriptionCrystal structures of RNA polymerase (RNAP) indicate that the RNAP β’ pincer (“clamp”) can exist in conformational states, ranging from a fully open conformation that permits entry and exit of DNA, to a fully closed conformation that prevents entry and exit of DNA. It has been hypothesized that the clamp also adopts multiple conformational states in solution and conformational changes in the clamp are important for function. In this work, a single-molecule fluorescence resonance energy transfer (smFRET) approach was developed that enables determination of RNAPclamp conformation in solution. smFRET was measured between a probe at the tip of the RNAP clamp and a probe at a fixed reference point in RNAP. A computational framework was then employed to interpret measured FRET efficiencies in terms of structural changes. Using this approach, RNAP clamp conformation was defined in each step of 70-dependent transcription initiation and elongation and in each step in σ54-dependent transcription initiation. Additionally, effects of four RNAP inhibitors, myxopyronin, corallopyronin, ripostatin and Gp2 on RNAP clamp conformation were assessed. It was observed that the clamp is predominantly open in free RNAP and in all steps leading up to the formation of a catalytically-competent-transcription-initiation complex. Upon formation of a catalytically-competent-transcription-initiation complex, the clamp closes, and continues to remain closed during transcription elongation. It was further observed that myxopyronin, corallopyronin, ripostatin and Gp2, prevent opening of the RNAP clamp. The results lead to the proposal that, the open clamp state is important for entry of DNA into, and unwinding of DNA in, the RNAP active center cleft during formation of a catalytically-competent-transcription initiation complex. The results lead to the proposal that, after entry of DNA into the RNAP active-center cleft upon formation of the catalytically competent transcription initiation complex, electrostatic interactions between the negatively charged DNA and the positively charged inner facet of the clamp, induce and/or stabilize clamp closure. The results are in agreement with the proposal that, clamp closure is important for stability of the catalytically competent transcription initiation complex and for stability and processivity of the transcription elongation complex.
NotePh.D.
NoteIncludes bibliographical references
Noteby Anirban Chakraborty
Genretheses, ETD doctoral
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.
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