Staff View
Ensemble fluorescence resonance energy transfer analysis of RNA polymerase clamp conformation

Descriptive

TypeOfResource
Text
TitleInfo (ID = T-1)
Title
Ensemble fluorescence resonance energy transfer analysis of RNA polymerase clamp conformation
SubTitle
PartName
PartNumber
NonSort
Identifier
ETD_1165
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000050468
Language (objectPart = )
LanguageTerm (authority = ISO639-2); (type = code)
eng
Genre (authority = marcgt)
theses
Subject (ID = SBJ-1); (authority = RUETD)
Topic
Chemistry and Chemical Biology
Subject (ID = SBJ-2); (authority = ETD-LCSH)
Topic
RNA polymerases
Subject (ID = SBJ-3); (authority = ETD-LCSH)
Topic
Fluorescence spectroscopy
Subject (ID = SBJ-4); (authority = ETD-LCSH)
Topic
Energy transfer
Abstract
Crystal 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.
PhysicalDescription
Form (authority = gmd)
electronic resource
Extent
xvi, 143 p. : ill.
InternetMediaType
application/pdf
InternetMediaType
text/xml
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references (p. 133-142)
Note (type = statement of responsibility)
by Dongye Wang
Name (ID = NAME-1); (type = personal)
NamePart (type = family)
Wang
NamePart (type = given)
Dongye
Role
RoleTerm (authority = RULIB); (type = )
author
DisplayForm
Dongye Wang
Name (ID = NAME-2); (type = personal)
NamePart (type = family)
Ebright
NamePart (type = given)
Richard
Role
RoleTerm (authority = RULIB); (type = )
chair
Affiliation
Advisory Committee
DisplayForm
Richard H Ebright
Name (ID = NAME-3); (type = personal)
NamePart (type = family)
Arnold
NamePart (type = given)
Eddy
Role
RoleTerm (authority = RULIB); (type = )
internal member
Affiliation
Advisory Committee
DisplayForm
Eddy Arnold
Name (ID = NAME-4); (type = personal)
NamePart (type = family)
Ludescher
NamePart (type = given)
Richard
Role
RoleTerm (authority = RULIB); (type = )
internal member
Affiliation
Advisory Committee
DisplayForm
Richard Ludescher
Name (ID = NAME-5); (type = personal)
NamePart (type = family)
Olson
NamePart (type = given)
Wilma
Role
RoleTerm (authority = RULIB); (type = )
internal member
Affiliation
Advisory Committee
DisplayForm
Wilma K Olson
Name (ID = NAME-6); (type = personal)
NamePart (type = family)
Nickels
NamePart (type = given)
Bryce
Role
RoleTerm (authority = RULIB); (type = )
outside member
Affiliation
Advisory Committee
DisplayForm
Bryce Nickels
Name (ID = NAME-1); (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB); (type = )
degree grantor
Name (ID = NAME-2); (type = corporate)
NamePart
Graduate School - New Brunswick
Role
RoleTerm (authority = RULIB); (type = )
school
OriginInfo
DateCreated (point = ); (qualifier = exact)
2008
DateOther (qualifier = exact); (type = degree)
2008-10
Place
PlaceTerm (type = code)
xx
Location
PhysicalLocation (authority = marcorg)
NjNbRU
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Identifier (type = doi)
doi:10.7282/T3WS8TJ7
Genre (authority = ExL-Esploro)
ETD doctoral
Back to the top

Rights

RightsDeclaration (AUTHORITY = GS); (ID = rulibRdec0006)
The author owns the copyright to this work.
Copyright
Status
Copyright protected
Availability
Status
Open
RightsEvent (AUTHORITY = rulib); (ID = 1)
Type
Permission or license
Detail
Non-exclusive ETD license
AssociatedObject (AUTHORITY = rulib); (ID = 1)
Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
Back to the top

Technical

ContentModel
ETD
MimeType (TYPE = file)
application/pdf
MimeType (TYPE = container)
application/x-tar
FileSize (UNIT = bytes)
4341760
Checksum (METHOD = SHA1)
3b28a43dc2194c5f30d08687cb5cb491ab072a08
Back to the top
Version 8.5.5
Rutgers University Libraries - Copyright ©2024