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Richard L. Gourse - One of the best experts on this subject based on the ideXlab platform.
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sequence independent upstream dna αctd interactions strongly stimulate escherichia coli rna polymerase LacUV5 promoter association
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Wilma Ross, Richard L. GourseAbstract:The C-terminal domains of the two α-subunits (αCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–αCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–αCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of αCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of αCTD decreased the composite second-order association rate constant, ka, of RNAP for the LacUV5 promoter by ≈10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k2. Removal of promoter DNA upstream of the -35 element also decreased both ka and k2 ≈10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with LacUV5 promoter fragments. The αCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with αCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the α-binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–αCTD interactions play an even larger role in transcription initiation than appreciated previously.
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Sequence-independent upstream DNA–αCTD interactions strongly stimulate Escherichia coli RNA polymerase-LacUV5 promoter association
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Wilma Ross, Richard L. GourseAbstract:The C-terminal domains of the two α-subunits (αCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–αCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–αCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of αCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of αCTD decreased the composite second-order association rate constant, ka, of RNAP for the LacUV5 promoter by ≈10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k2. Removal of promoter DNA upstream of the -35 element also decreased both ka and k2 ≈10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with LacUV5 promoter fragments. The αCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with αCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the α-binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–αCTD interactions play an even larger role in transcription initiation than appreciated previously.
Wilma Ross - One of the best experts on this subject based on the ideXlab platform.
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sequence independent upstream dna αctd interactions strongly stimulate escherichia coli rna polymerase LacUV5 promoter association
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Wilma Ross, Richard L. GourseAbstract:The C-terminal domains of the two α-subunits (αCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–αCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–αCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of αCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of αCTD decreased the composite second-order association rate constant, ka, of RNAP for the LacUV5 promoter by ≈10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k2. Removal of promoter DNA upstream of the -35 element also decreased both ka and k2 ≈10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with LacUV5 promoter fragments. The αCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with αCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the α-binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–αCTD interactions play an even larger role in transcription initiation than appreciated previously.
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Sequence-independent upstream DNA–αCTD interactions strongly stimulate Escherichia coli RNA polymerase-LacUV5 promoter association
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Wilma Ross, Richard L. GourseAbstract:The C-terminal domains of the two α-subunits (αCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA–αCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA–αCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of αCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of αCTD decreased the composite second-order association rate constant, ka, of RNAP for the LacUV5 promoter by ≈10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k2. Removal of promoter DNA upstream of the -35 element also decreased both ka and k2 ≈10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with LacUV5 promoter fragments. The αCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with αCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the α-binding sites. In addition to highlighting the functional importance of nonspecific protein–DNA interactions, these results suggest also that UP element–αCTD interactions play an even larger role in transcription initiation than appreciated previously.
Konstantin Brodolin - One of the best experts on this subject based on the ideXlab platform.
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The sigma 70 subunit of RNA polymerase induces LacUV5 promoter-proximal pausing of transcription.
Nature structural & molecular biology, 2004Co-Authors: Konstantin Brodolin, Nikolay Zenkin, Arkady Mustaev, Daria Mamaeva, Hermann HeumannAbstract:The σ70 subunit of Escherichia coli RNA polymerase (RNAP) is a transcription initiation factor that can also be associated with RNAP during elongation. We provide biochemical evidence that σ70 induces a transcription pause at the LacUV5 promoter after RNAP has synthesized a 17-nucleotide transcript. The σ70-dependent pausing requires an interaction between σ70 and a part of the lac repressor operator sequence resembling a promoter −10 consensus. The polysaccharide heparin triggers the release of σ70 from the paused complexes, supporting the view that during the transition from initiation to elongation the interactions between σ70 and core RNAP are weakened. We propose that the binding and retention of σ70 in elongation complexes are stabilized by its ability to form contacts with DNA of the transcription bubble. In addition, we suggest that the σ70 subunit in the elongation complex may provide a target for regulation of gene expression.
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Topography of LacUV5 initiation complexes.
Nucleic acids research, 2001Co-Authors: Vasily M. Studitsky, Konstantin Brodolin, Ye Liu, Andrei D. MirzabekovAbstract:Formation of a transcriptionally competent open complex is a highly regulated multistep process involving at least two intermediates. The rate of formation and stability of the intermediate complexes often determine promoter strength. However, the detailed mechanism of formation of the open complex and the high resolution structures of these intermediates are not known. In this study the structures of the open and intermediate complexes formed on the LacUV5 promoter by Escherichia coli RNA polymerase were analyzed using 'zero length' DNA-protein cross-linking. In both the open and the intermediate complexes the core subunits (ss' and ss) interact with LacUV5 DNA in a similar way, forming DNA-protein contacts flanking the initiation site. At the same time, the recognition (sigma(70)) subunit closely interacts with the promoter only in the open complex. In combination with our previous results, the data suggest that during promoter recognition contacts of the sigma subunit with core RNA polymerase and promoter DNA are rearranged in concert. These rearrangements constitute a landmark of transition from the intermediate to the open complex, identifying the sigma subunit as a key player directing formation of the open complex.
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Identification of RNA Polymerase β′ Subunit Segment Contacting the Melted Region of the LacUV5 Promoter
The Journal of biological chemistry, 2000Co-Authors: Konstantin Brodolin, Arkady Mustaev, Konstantin Severinov, Vadim NikiforovAbstract:Abstract Identification of the RNA polymerase functional regions involved in interactions with promoter is a basis for understanding the mechanism of transcription initiation. We have used formaldehyde cross-linking to identify a region of Escherichia coli RNA polymerase β′ subunit contacting LacUV5 promoter in open complex. Treatment of open complex with formaldehyde results in cross-linking of β′ and ς70 subunits at positions −5 and −3 on the nontemplate strand of the promoter DNA. These cross-links reflect specific interactions between RNA polymerase and promoter established in open complex. The positions of formaldehyde cross-links in the β′ subunit were mapped to the N-terminal segment (Cys198–Met237), which is contiguous to the evolutionary conserved region B. The proximity of the β′ and ς cross-links suggest that the N-terminal region of the β′ subunit, interacting with single-stranded promoter DNA, can cooperate with the ς subunit in the process of open complex formation.
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Conformational changes in E.coli RNA polymerase during promoter recognition
Nucleic acids research, 1993Co-Authors: Konstantin Brodolin, Vasily M. Studitsky, Andrei D. MirzabekovAbstract:We analysed complexes formed during recognition of the LacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions. In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes. In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.
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Study of the structure of Escherichia coli RNA polymerase and its complex with the LacUV5-promotor using protein-protein and DNA-protein crosslinks, formed by formaldehyde
Molekuliarnaia biologiia, 1993Co-Authors: Konstantin Brodolin, Andrei D. MirzabekovAbstract:The protein-protein and DNA-protein crosslinks produced by formaldehyde were used to investigate the intersubunit and subunit-DNA interactions for free RNA polymerase and for an open complex of RNA polymerase with the LacUV5 promoter. In both cases the contacts between beta,beta' and beta', sigma subunits were observed, while there were no contacts between beta and sigma subunits. Only one of beta or beta' subunits and a sigma subunit crosslink to promoter DNA. We have chosen the conditions for fixing the RNA polymerase-DNA complexes on different stages of transcription initiation. The possibility to use limited fixation with low concentrations of formaldehyde to study specific DNA-protein interactions was shown.
Malcolm Buckle - One of the best experts on this subject based on the ideXlab platform.
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FIS modulates the kinetics of successive interactions of RNA polymerase with the core and upstream regions of the tyrT promoter.
Journal of Molecular Biology, 2002Co-Authors: Iain Pemberton, Georgi Muskhelishvili, Andrew A Travers, Malcolm BuckleAbstract:We have applied laser UV photo-footprinting to characterise kinetically complexes involving the activator protein FIS, RNA polymerase and the tyrT promoter of Escherichia coli. FIS photo-footprints strongly to three binding sites upstream of the core promoter. The polymerase photo-footprints in the near-consensus -35 hexamer on the non-template strand of DNA in a fashion similar to that of stable complexes involving the LacUV5 promoter. The kinetics of the interactions of polymerase alone with the tyrT promoter differ from those observed previously at the LacUV5 promoter. In the absence of FIS, we observe an upstream polymerase-induced signal at -122 within FIS site III that occurs subsequent to changes in the core promoter region and is strongly dependent on negative supercoiling. These observations support the proposal that the upstream region of the promoter is wrapped around the polymerase. We propose that the wrapped DNA allows the polymerase to overcome, at least in part, the barrier to DNA untwisting imparted by the G+C-rich discriminator. We further suggest that FIS plays a similar role and may facilitate polymerase escape.
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Differential melting of the transcription start site associated with changes in RNA polymerase-promoter contacts in initiating transcription complexes.
Journal of Molecular Biology, 2001Co-Authors: K. Brodolin, Malcolm BuckleAbstract:Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-LacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.
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E.coli RNApolymerase, deleted intheC-terminal part of its a-subunit, interacts differently withthecAMP-CRP complex atthelacPl andatthega/Pl promoter
1993Co-Authors: Annie Kolb, Kazuhiko Igarashi, Malcolm BuckleAbstract:Adeletion oftheC-terminal part ofthea-subunit ofRNA polymerase isknowntoaffect differently promoters activated byCRPdepending onthelocation oftheCRP binding site atthepromoter. WhentheCRPbinding site islocated at-61.5, asatlacPl (atypeIpromoter), activation isstrongly Impaired whileitisnot significantly affected atgaIPl whereCRPbinds 41.5 bpupstream ofthestart ofthemessage(type 11 promoter). Wehaveinvestigated thedifferences Inthe architecture ofthecorresponding opencomplexes by comparingthe positioning of holoenzymes reconstituted respectively withnative orwithtruncated c-subunits (containing thefirst 235or256residues of a)attwo'up' promoter mutants ofthelacPl andgaIP1 promoters (respectively LacUV5 andgaI9Al 6C).First, theaffinity ofwild-type RNA polymerase forboth promoters isincreased bythepresence ofCRPand cAMP.Bycontrast, holoenzymes reconstituted with truncated a-subunits, showcooperative binding atthe gaIPl promoter only. Second, footprinting dataconfirm theseobservations andindicate thatthetruncated holoenzymes areunable torecognize regions ofthe promoter upstream fromposition -40.Theabsence ofcontacts between thetruncated enzymes andCRP atthelacPl promoter canexplain thedeficiency in activation. AtthegaIP1 promoter, wheretheCRPsite iscloser totheinitiation site, protein-protein contacts canstill occur withthetruncated polymerases, showing that theC-terminal part oftheat-subunit isnotinvolved
Andrei D. Mirzabekov - One of the best experts on this subject based on the ideXlab platform.
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Topography of LacUV5 initiation complexes.
Nucleic acids research, 2001Co-Authors: Vasily M. Studitsky, Konstantin Brodolin, Ye Liu, Andrei D. MirzabekovAbstract:Formation of a transcriptionally competent open complex is a highly regulated multistep process involving at least two intermediates. The rate of formation and stability of the intermediate complexes often determine promoter strength. However, the detailed mechanism of formation of the open complex and the high resolution structures of these intermediates are not known. In this study the structures of the open and intermediate complexes formed on the LacUV5 promoter by Escherichia coli RNA polymerase were analyzed using 'zero length' DNA-protein cross-linking. In both the open and the intermediate complexes the core subunits (ss' and ss) interact with LacUV5 DNA in a similar way, forming DNA-protein contacts flanking the initiation site. At the same time, the recognition (sigma(70)) subunit closely interacts with the promoter only in the open complex. In combination with our previous results, the data suggest that during promoter recognition contacts of the sigma subunit with core RNA polymerase and promoter DNA are rearranged in concert. These rearrangements constitute a landmark of transition from the intermediate to the open complex, identifying the sigma subunit as a key player directing formation of the open complex.
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Conformational changes in E.coli RNA polymerase during promoter recognition
Nucleic acids research, 1993Co-Authors: Konstantin Brodolin, Vasily M. Studitsky, Andrei D. MirzabekovAbstract:We analysed complexes formed during recognition of the LacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21. The protein-protein cross-linking pattern of contacting subunits is the same for the RNA polymerase in solution and in RPO: there are strong sigma-beta' and beta-beta' interactions. In contrast, only beta-beta' cross-links were detected in the closed (RPC) and intermediate (RPI) complexes. In presence of lac repressor before or after formation of the RPO cross-linking pattern is similar with that of RPI (RPC) complex.
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Study of the structure of Escherichia coli RNA polymerase and its complex with the LacUV5-promotor using protein-protein and DNA-protein crosslinks, formed by formaldehyde
Molekuliarnaia biologiia, 1993Co-Authors: Konstantin Brodolin, Andrei D. MirzabekovAbstract:The protein-protein and DNA-protein crosslinks produced by formaldehyde were used to investigate the intersubunit and subunit-DNA interactions for free RNA polymerase and for an open complex of RNA polymerase with the LacUV5 promoter. In both cases the contacts between beta,beta' and beta', sigma subunits were observed, while there were no contacts between beta and sigma subunits. Only one of beta or beta' subunits and a sigma subunit crosslink to promoter DNA. We have chosen the conditions for fixing the RNA polymerase-DNA complexes on different stages of transcription initiation. The possibility to use limited fixation with low concentrations of formaldehyde to study specific DNA-protein interactions was shown.
