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TitleEnsemble fluorescence resonance energy transfer analysis of RNA polymerase clamp conformation
NameWang, Dongye (author), Ebright, Richard (chair), Arnold, Eddy (internal member), Ludescher, Richard (internal member), Olson, Wilma (internal member), Nickels, Bryce (outside member), Rutgers University, Graduate School - New Brunswick,
Degree Date2008-10
Date Created2008
SubjectChemistry and Chemical Biology,
RNA polymerases,
Fluorescence spectroscopy,
Energy transfer
DescriptionCrystal structures of RNA Polymerase (RNAP) and RNAP complexes indicate that the RNAP beta' pincer ("clamp") can exist in a range of 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. Clamp closure involves a swinging motion of the beta' pincer by the switch region at the base of the beta' pincer.
In order to define RNAP clamp conformation in solution, we have used fluorescence resonance energy transfer (FRET) to monitor the distance between a first fluorescent probe, serving as donor, incorporated at the tip of the beta' pincer and a second fluorescent probe, serving as acceptor, incorporated at the tip of the beta pincer.
We have developed a procedure that permits incorporation of a fluorescent probe within a protein. The procedure involves preparation of a protein containing the azide-containing unnatural amino acid p-azidophenylalanine at the site of interest, followed by incorporation of a fluorescent probe through azide-specific chemical modification (accomplished by Staudinger-Bertozzi ligation using a phosphine derivative of the fluorescent probe). We have used this procedure to incorporate fluorescent probes at the tips of the RNAP beta' pincer and RNAP beta' pincer.
We have used the resulting labeled RNAP derivatives in FRET experiments addressing opening and closing of the RNAP active-center-cleft in transcription initiation and elongation and in FRET experiments addressing effects of small-molecule effectors, myxopyronin (Myx), corallopyronin (Cor), ripostatin (Rip), and lipiarmycin (Lpm), on opening and closing of the RNAP active-center cleft. Results indicate that: (1) RNAP holoenzyme in solution exists predominantly in a partly closed clamp conformational state (2) the RNAP clamp closes upon formation of the RNAP-promoter open complex, yielding a fully closed clamp conformational state (3) the RNAP clamp remains closed--and exhibits no further change in mean clamp conformation--upon formation of RNAP-promoter initial transcribing complexes and transcription elongation complexes. The results support the proposal that Myx, Cor, Rip and Lpm bind to an RNAP- switch-region conformational state and Myx. Cor, Rip and Lpm inhibit RNAP function by trapping the RNAP switch region in this conformational state, thereby interfering with conformational cycling of RNAP clamp important for RNAP function.
In order to define RNAP clamp conformation in solution, we have used fluorescence resonance energy transfer (FRET) to monitor the distance between a first fluorescent probe, serving as donor, incorporated at the tip of the beta' pincer and a second fluorescent probe, serving as acceptor, incorporated at the tip of the beta pincer.
We have developed a procedure that permits incorporation of a fluorescent probe within a protein. The procedure involves preparation of a protein containing the azide-containing unnatural amino acid p-azidophenylalanine at the site of interest, followed by incorporation of a fluorescent probe through azide-specific chemical modification (accomplished by Staudinger-Bertozzi ligation using a phosphine derivative of the fluorescent probe). We have used this procedure to incorporate fluorescent probes at the tips of the RNAP beta' pincer and RNAP beta' pincer.
We have used the resulting labeled RNAP derivatives in FRET experiments addressing opening and closing of the RNAP active-center-cleft in transcription initiation and elongation and in FRET experiments addressing effects of small-molecule effectors, myxopyronin (Myx), corallopyronin (Cor), ripostatin (Rip), and lipiarmycin (Lpm), on opening and closing of the RNAP active-center cleft. Results indicate that: (1) RNAP holoenzyme in solution exists predominantly in a partly closed clamp conformational state (2) the RNAP clamp closes upon formation of the RNAP-promoter open complex, yielding a fully closed clamp conformational state (3) the RNAP clamp remains closed--and exhibits no further change in mean clamp conformation--upon formation of RNAP-promoter initial transcribing complexes and transcription elongation complexes. The results support the proposal that Myx, Cor, Rip and Lpm bind to an RNAP- switch-region conformational state and Myx. Cor, Rip and Lpm inhibit RNAP function by trapping the RNAP switch region in this conformational state, thereby interfering with conformational cycling of RNAP clamp important for RNAP function.
NotePh.D.
NoteIncludes bibliographical references (p. 133-142)
Noteby Dongye Wang
Genretheses
Persistent URLhttp://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000050468
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization Name
RightsThe author owns the copyright to this work.