The Experts below are selected from a list of 1107 Experts worldwide ranked by ideXlab platform
Richard H. Ebright - One of the best experts on this subject based on the ideXlab platform.
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The mechanism of Transcription start site selection
2017Co-Authors: Jared T. Winkelman, Bryce E. Nickels, Terence R. Strick, Chirangini Pukhrambam, Richard H. EbrightAbstract:During Transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded “Transcription Bubble,” and selects a Transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA “scrunching” and “anti-scrunching,” the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the Transcription Bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.
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Interactions between RNA polymerase and the core recognition element are a determinant of Transcription start site selection
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Irina O. Vvedenskaya, Richard H. Ebright, Hanif Vahedian-movahed, Yuanchao Zhang, Deanne Taylor, Bryce E. NickelsAbstract:During Transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded Transcription Bubble, and selects a template-strand nucleotide to serve as the Transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the Transcription Bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the Transcription Bubble in RPo, and thereby modulate TSS selection, which involves Transcription Bubble expansion or Transcription Bubble contraction (scrunching or antiscrunching).
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massively systematic transcript end readout master Transcription start site selection Transcriptional slippage and transcript yields
Molecular Cell, 2015Co-Authors: Irina O. Vvedenskaya, Richard H. Ebright, Yuanchao Zhang, Deanne Taylor, Seth R Goldman, Anna Valenti, Valeria Visone, Bryce E. NickelsAbstract:Summary We report the development of a next-generation sequencing-based technology that entails construction of a DNA library comprising up to at least 4 7 (∼16,000) barcoded sequences, production of RNA transcripts, and analysis of transcript ends and transcript yields (massively systematic transcript end readout, "MASTER"). Using MASTER, we define full inventories of Transcription start sites ("TSSomes") of Escherichia coli RNA polymerase for initiation at a consensus core promoter in vitro and in vivo; we define the TSS-region DNA sequence determinants for TSS selection, reiterative initiation ("slippage synthesis"), and transcript yield; and we define effects of DNA topology and NTP concentration. The results reveal that slippage synthesis occurs from the majority of TSS-region DNA sequences and that TSS-region DNA sequences have profound, up to 100-fold, effects on transcript yield. The results further reveal that TSSomes depend on DNA topology, consistent with the proposal that TSS selection involves Transcription-Bubble expansion ("scrunching") and Transcription-Bubble contraction ("anti-scrunching").
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the Transcription Bubble of the rna polymerase promoter open complex exhibits conformational heterogeneity and millisecond scale dynamics implications for Transcription start site selection
Journal of Molecular Biology, 2013Co-Authors: Nicole C. Robb, Richard H. Ebright, Thorben Cordes, Ling Chin Hwang, Kristofer Gryte, Diego Duchi, Timothy D. Craggs, Yusdi Santoso, Shimon WeissAbstract:Bacterial Transcription is initiated after RNA polymerase (RNAP) binds to promoter DNA, melts ~14 bp around the Transcription start site and forms a single-stranded “Transcription Bubble” within a catalytically active RNAP–DNA open complex (RPo). There is significant flexibility in the Transcription start site, which causes variable spacing between the promoter elements and the start site; this in turn causes differences in the length and sequence at the 5′ end of RNA transcripts and can be important for gene regulation. The start-site variability also implies the presence of some flexibility in the positioning of the DNA relative to the RNAP active site in RPo. The flexibility may occur in the positioning of the Transcription Bubble prior to RNA synthesis and may reflect Bubble expansion (“scrunching”) or Bubble contraction (“unscrunching”). Here, we assess the presence of dynamic flexibility in RPo with single-molecule FRET (Forster resonance energy transfer). We obtain experimental evidence for dynamic flexibility in RPo using different FRET rulers and labeling positions. An analysis of FRET distributions of RPo using burst variance analysis reveals conformational fluctuations in RPo in the millisecond timescale. Further experiments using subsets of nucleotides and DNA mutations allowed us to reprogram the Transcription start sites, in a way that can be described by repositioning of the single-stranded Transcription Bubble relative to the RNAP active site within RPo. Our study marks the first experimental observation of conformational dynamics in the Transcription Bubble of RPo and indicates that DNA dynamics within the Bubble affect the search for Transcription start sites.
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Flexibility in Transcription Start-Site Selection by RNA Polymerase Involves Transcription-Bubble Expansion (“Scrunching”) or Contraction (“Unscrunching”)
Biophysical Journal, 2013Co-Authors: Mathivanan Chinnaraj, Terence R. Strick, Richard H. EbrightAbstract:RNA polymerase (RNAP) is a molecular machine that carries out a series of reactions during Transcription initiation:(i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex (RPc).(ii) RNAP unwinds ∼13 base pairs of promoter DNA surrounding the Transcription start site, forming a single-stranded region ("Transcription Bubble"), and yielding an RNAP-promoter open complex (RPo).(iii) RNAP begins synthesis of an RNA product as an RNAP-promoter initial transcribing complex (RPitc).(iv) After RNAP synthesizes an RNA product ∼11 nt in length, RNAP breaks its interactions with the promoter, escapes from the promoter, and begins Transcription elongation as an RNAP-DNA elongation complex (RDe).It has been known for four decades that the Transcription start site can vary over a range of at least 5 bp--comprising the default start site (position +1), downstream-shifted start sites, (positions +2 and +3), and upstream-sifted start sites (positions −2 and −1)--and that the Transcription start site can be re-programmed within this range by the use of appropriate ribodinucleotide primers. However, the mechanistic basis of this flexibility in Transcription start-site selection has not been known.In this work, we have used magnetic-tweezers single-molecule nanomanipulation to monitor the extent of RNAP-dependent DNA unwinding in Transcription initiation complexes containing ribodinucleotide primers that re-program Transcription to start at downstream-shifted start sites (positions +2 or +3) or upstream-shifted start sites (positions −2 or −1). The results indicate that re-programming the Transcription start site changes the Transcription-Bubble size: forcing a downstream-shifted start site increases Transcription-Bubble size, and forcing upstream-shifted start sites decreases Transcription-Bubble size. The results support a model in which flexibility in Transcription start-site selection is a consequence of pre-initiation Transcription-Bubble expansion (“pre-initiation scrunching”) or pre-initiation Transcription-Bubble contraction (“pre-initiation unscrunching”).
Terence R. Strick - One of the best experts on this subject based on the ideXlab platform.
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Single-molecule characterization of extrinsic Transcription termination by Sen1 helicase.
Nature communications, 2019Co-Authors: S. Wang, Z. Han, D. Libri, O. Porrua, Terence R. StrickAbstract:Extrinsic Transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate Transcription without Transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II Transcription Bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion. Yeast’s Sen1 helicase is involved in the suppression of antisense Transcription from bidirectional eukaryotic promoters. Here authors develop and utilize a quantitative single-molecule assay reporting on the kinetics of extrinsic eukaryotic Transcription termination by the Sen1 helicase and a reaction intermediate in which the Pol II Transcription Bubble appears half-rewound.
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The mechanism of Transcription start site selection
2017Co-Authors: Jared T. Winkelman, Bryce E. Nickels, Terence R. Strick, Chirangini Pukhrambam, Richard H. EbrightAbstract:During Transcription initiation, RNA polymerase (RNAP) binds to promoter DNA, unwinds promoter DNA to form an RNAP-promoter open complex (RPo) containing a single-stranded “Transcription Bubble,” and selects a Transcription start site (TSS). TSS selection occurs at different positions within the promoter region, depending on promoter sequence and initiating-substrate concentration. Variability in TSS selection has been proposed to involve DNA “scrunching” and “anti-scrunching,” the hallmarks of which are: (i) forward and reverse movement of the RNAP leading edge, but not trailing edge, relative to DNA, and (ii) expansion and contraction of the Transcription Bubble. Here, using in vitro and in vivo protein-DNA photocrosslinking and single-molecule nanomanipulation, we show bacterial TSS selection exhibits both hallmarks of scrunching and anti-scrunching, and we define energetics of scrunching and anti-scrunching. The results establish the mechanism of TSS selection by bacterial RNAP and suggest a general mechanism for TSS selection by bacterial, archaeal, and eukaryotic RNAP.
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Correlative Nanomanipulation and Colocalization of Single-Molecules to Study Transcription-Coupled DNA Repair
Biophysical Journal, 2015Co-Authors: Evan T Graves, Camille Duboc, Jun Fan, Terence R. StrickAbstract:In Transcription-coupled repair (TCR), E. coli RNA polymerase (RNAP) stalled on a DNA lesion is removed by the Mfd tanslocase, which then recruits downstream repair components UvrA/UvrB. Mfd-RNAP interactions can be observed in real-time using the magnetic trapping of single DNA molecules to monitor states of the Transcription Bubble created by RNA polymerase. Remodelling of the stalled RNAP Transcription Bubble occurs in a sequence of two mechanical steps with formation of a long-lived intermediate. In the intermediate state, mechanical signatures of protein-DNA interactions are insufficient to clearly identify which protein partners are interacting with the DNA, as they could involve RNAP, Mfd, or a combination of the two. We combine single-molecule nanomanipulation and single-molecule fluorescence in a TIRF field to monitor, in real-time, the arrival and departure of the different components of the reaction, via fluorescence, while we simultaneously record the chemo-mechanical state of the protein-DNA complex, via nanomanipulation. This allows us to correlate the catalytic state to the molecular composition of this dynamic, multicomponent DNA repair complex.
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Flexibility in Transcription Start-Site Selection by RNA Polymerase Involves Transcription-Bubble Expansion (“Scrunching”) or Contraction (“Unscrunching”)
Biophysical Journal, 2013Co-Authors: Mathivanan Chinnaraj, Terence R. Strick, Richard H. EbrightAbstract:RNA polymerase (RNAP) is a molecular machine that carries out a series of reactions during Transcription initiation:(i) RNAP binds to promoter DNA, yielding an RNAP-promoter closed complex (RPc).(ii) RNAP unwinds ∼13 base pairs of promoter DNA surrounding the Transcription start site, forming a single-stranded region ("Transcription Bubble"), and yielding an RNAP-promoter open complex (RPo).(iii) RNAP begins synthesis of an RNA product as an RNAP-promoter initial transcribing complex (RPitc).(iv) After RNAP synthesizes an RNA product ∼11 nt in length, RNAP breaks its interactions with the promoter, escapes from the promoter, and begins Transcription elongation as an RNAP-DNA elongation complex (RDe).It has been known for four decades that the Transcription start site can vary over a range of at least 5 bp--comprising the default start site (position +1), downstream-shifted start sites, (positions +2 and +3), and upstream-sifted start sites (positions −2 and −1)--and that the Transcription start site can be re-programmed within this range by the use of appropriate ribodinucleotide primers. However, the mechanistic basis of this flexibility in Transcription start-site selection has not been known.In this work, we have used magnetic-tweezers single-molecule nanomanipulation to monitor the extent of RNAP-dependent DNA unwinding in Transcription initiation complexes containing ribodinucleotide primers that re-program Transcription to start at downstream-shifted start sites (positions +2 or +3) or upstream-shifted start sites (positions −2 or −1). The results indicate that re-programming the Transcription start site changes the Transcription-Bubble size: forcing a downstream-shifted start site increases Transcription-Bubble size, and forcing upstream-shifted start sites decreases Transcription-Bubble size. The results support a model in which flexibility in Transcription start-site selection is a consequence of pre-initiation Transcription-Bubble expansion (“pre-initiation scrunching”) or pre-initiation Transcription-Bubble contraction (“pre-initiation unscrunching”).
Rui J Sousa - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of T7 RNAP Pausing and Termination at the T7 Concatemer Junction: A Local Change in Transcription Bubble Structure Drives a Large Change in Transcription Complex Architecture
Journal of molecular biology, 2007Co-Authors: Dhananjaya Nayak, Sylvester Siller, Qing Guo, Rui J SousaAbstract:The T7RNA polymerase (RNAP) elongation complex (EC) pauses and is destabilized at a unique 8 nucleotide (nt) sequence found at the junction of the head-to-tail concatemers of T7 genomic DNA generated during T7 DNA replication. The paused EC may recruit the T7 DNA processing machinery, which cleaves the concatemerized DNA within this 8 nt concatemer junction (CJ). Pausing of the EC at the CJ involves structural changes in both the RNAP and Transcription Bubble. However, these structural changes have not been fully defined, nor is it understood how the CJ sequence itself causes the EC to change its structure, to pause, and to become less stable. Here we use solution and RNAP-tethered chemical nucleases to probe the CJ transcript and changes in the EC structure as the polymerase pauses and terminates at the CJ. Together with extensive mutational scanning of regions of the polymerase that are likely to be involved in recognition of the CJ, we are able to develop a description of the events that occur as the EC transcribes through the CJ and subsequently pauses. In this process, a local change in the structure of the Transcription Bubble drives a large change in the architecture of the EC. This altered EC structure may then serve as the signal that recruits the processing machinery to the CJ.
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Functional architecture of T7 RNA polymerase Transcription complexes.
Journal of molecular biology, 2007Co-Authors: Dhananjaya Nayak, Qing Guo, Rui J SousaAbstract:Bacteriophage T7 RNA polymerase is the best-characterized member of a widespread family of single-subunit RNA polymerases. Crystal structures of T7 RNA polymerase initiation and elongation complexes have provided a wealth of detailed information on RNA polymerase interactions with the promoter and Transcription Bubble, but the absence of DNA downstream of the melted region of the template in the initiation complex structure, and the absence of DNA upstream of the Transcription Bubble in the elongation complex structure means that our picture of the functional architecture of T7 RNA polymerase Transcription complexes remains incomplete. Here, we use the site-specifically tethered chemical nucleases and functional characterization of directed T7 RNAP mutants to both reveal the architecture of the duplex DNA that flanks the Transcription Bubble in the T7 RNAP initiation and elongation complexes, and to define the function of the interactions made by these duplex elements. We find that downstream duplex interactions made with a cluster of lysine residues (K711/K713/K714) are present during both elongation and initiation, where they contribute to stabilizing a bend in the downstream DNA that is important for promoter opening. The upstream DNA in the elongation complex is also found to be sharply bent at the upstream edge of the Transcription Bubble, thereby allowing formation of upstream duplex:polymerase interactions that contribute to elongation complex stability.
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The T7 RNA Polymerase Intercalating Hairpin Is Important for Promoter Opening during Initiation but Not for RNA Displacement or Transcription Bubble Stability during Elongation
Biochemistry, 2001Co-Authors: Luis G. Brieba, Rui J SousaAbstract:The recently described crystal structures of a T7RNAP−promoter complex and an initial Transcription complex reveal a β-hairpin which inserts between the template and nontemplate strands of the promoter [Cheetham, G. M., et al. (1999) Nature 399, 80; Cheetham, G. M., et al. (1999) Science 286, 2305]. A stacking interaction between the exposed DNA bases and a valine at the tip of this hairpin may be especially important for stabilizing the opened promoter during initiation. It has been suggested that this hairpin may also be important for holding the Transcription Bubble open during transcript elongation, and a proposed model for how the RNA exits the Transcription complex implies that this hairpin may also help displace the RNA from the template strand. To test these hypotheses, we have characterized both point and deletion mutants of this element. We find that these mutants exhibit reduced activity on linear, double-stranded templates but not on supercoiled or partially single-stranded templates. Probing ...
Konstantin Severinov - One of the best experts on this subject based on the ideXlab platform.
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coupling of downstream rna polymerase promoter interactions with formation of catalytically competent Transcription initiation complex
Journal of Molecular Biology, 2014Co-Authors: Vladimir Mekler, Arkady Mustaev, Leonid Minakhin, Konstantin Severinov, Sergei BorukhovAbstract:Abstract Bacterial RNA polymerase (RNAP) makes extensive contacts with duplex DNA downstream of the Transcription Bubble in initiation and elongation complexes. We investigated the role of downstream interactions in formation of catalytically competent Transcription initiation complex by measuring initiation activity of stable RNAP complexes with model promoter DNA fragments whose downstream ends extend from + 3 to + 21 relative to the Transcription start site at + 1. We found that DNA downstream of position + 6 does not play a significant role in Transcription initiation when RNAP–promoter interactions upstream of the Transcription start site are strong and promoter melting region is AT rich. Further shortening of downstream DNA dramatically reduces efficiency of Transcription initiation. The boundary of minimal downstream DNA duplex needed for efficient Transcription initiation shifted further away from the catalytic center upon increasing the GC content of promoter melting region or in the presence of bacterial stringent response regulators DksA and ppGpp. These results indicate that the strength of RNAP–downstream DNA interactions has to reach a certain threshold to retain the catalytically competent conformation of the initiation complex and that establishment of contacts between RNAP and downstream DNA can be coupled with promoter melting. The data further suggest that RNAP interactions with DNA immediately downstream of the Transcription Bubble are particularly important for initiation of Transcription. We hypothesize that these active center-proximal contacts stabilize the DNA template strand in the active center cleft and/or position the RNAP clamp domain to allow RNA synthesis.
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Quantitative Dissection of RNA Polymerase-Promoter Interactions using Protein Beacon Assay
Biophysical Journal, 2012Co-Authors: Vladimir Mekler, Leonid Minakhin, Olga Pavlova, Konstantin SeverinovAbstract:Promoter recognition and melting by RNA polymerase (RNAP) are key points in gene expression and regulation. E. coli RNAP binding to promoter DNA and model promoter fragments can be measured using a new protein beacon assay. The assay relies on the detection of fluorescence signal from a fluorescent label incorporated into the σ70 subunit of RNAP close to region 2.3 of σ70, part of RNAP that recognizes the −10 promoter element. The ground level fluorescence of such RNAP beacon is low because the region 2.3 aromatic amino acids quench the fluorescence. When RNAP beacon binds promoter, the quenching interactions become destroyed, leading to increased fluorescence.Promoter melting in bacteria is nucleated at upstream edge of the Transcription Bubble. The mechanism of downstream propagation of the Transcription Bubble to include the Transcription start site is unclear. We introduced new downstream fork junction promoter fragments mimicking the downstream segment of promoter complexes. We demonstrated that RNAP binding to downstream fork junctions was coupled with DNA melting around the Transcription start point and identified structural determinants of affinity and Transcription activity of RNAP-downstream fork junction complexes.The product of E. coli T7 bacteriophage gene 2 (gp2 protein) is a potent inhibitor of host RNAP. We applied the beacon assay to the mechanism of gp2 inhibition. We measured the effect of gp2 on RNAP binding to various promoter fragments. In this way, the effect of gp2 on RNAP-promoter interactions was dissected. Gp2 greatly decreased RNAP affinity to downstream promoter duplex and inhibited RNAP binding to template and non-template strand segments located between the −10 promoter element and downstream edge of the Transcription Bubble. The inhibition of RNAP interactions with the Transcription Bubble by gp2 is a novel effect, which may occur via allosteric mechanism.
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Molecular mechanism of Transcription inhibition by phage T7 gp2 protein
Journal of molecular biology, 2011Co-Authors: Vladimir Mekler, Leonid Minakhin, Carol Sheppard, Sivaramesh Wigneshweraraj, Konstantin SeverinovAbstract:Abstract Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β′ jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the Transcription start. As a result, the complex is Transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP–gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β′ jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the Transcription Bubble downstream of the − 10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the Transcription Bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β′ jaw.
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A Critical Role of Downstream RNA Polymerase-Promoter Interactions in the Formation of Initiation Complex
The Journal of biological chemistry, 2011Co-Authors: Vladimir Mekler, Leonid Minakhin, Konstantin SeverinovAbstract:Nucleation of promoter melting in bacteria is coupled with RNA polymerase (RNAP) binding to a conserved -10 promoter element located at the upstream edge of the Transcription Bubble. The mechanism of downstream propagation of the Transcription Bubble to include the Transcription start site is unclear. Here we introduce new model downstream fork junction promoter fragments that specifically bind RNAP and mimic the downstream segment of promoter complexes. We demonstrate that RNAP binding to downstream fork junctions is coupled with DNA melting around the Transcription start point. Consequently, certain downstream fork junction probes can serve as Transcription templates. Using a protein beacon fluorescent method, we identify structural determinants of affinity and Transcription activity of RNAP-downstream fork junction complexes. Measurements of RNAP interaction with double-stranded promoter fragments reveal that the strength of RNAP interactions with downstream DNA plays a critical role in promoter opening and that the length of the downstream duplex must exceed a critical length for efficient formation of Transcription competent open promoter complex.
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mapping of rna polymerase residues that interact with bacteriophage xp10 Transcription antitermination factor p7
Journal of Molecular Biology, 2008Co-Authors: Yulia Yuzenkova, Nikolay Zenkin, Konstantin SeverinovAbstract:Bacteriophage Xp10-encoded Transcription factor p7 interacts with host Xanthomonas oryzae RNA polymerase β′ subunit and prevents both promoter recognition by the RNA polymerase holoenzyme and Transcription termination by the RNA polymerase core. P7 does not bind to and has no effect on RNA polymerase from E. coli. Here, we use a combination of biochemical and genetic methods to map the p7 interaction site to within four β′ amino acids at the N-terminus of X. oryzae RNAP β′. The interaction site is located in an area that is close to the promoter spacer in the open complex and to the upstream boundary of the Transcription Bubble in the elongation complex, providing possible explanation as to how a small protein can affect both Transcription initiation and termination by binding to the same RNA polymerase site.
Jeffrey W Roberts - One of the best experts on this subject based on the ideXlab platform.
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role of dna Bubble rewinding in enzymatic Transcription termination
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Jooseop Park, Jeffrey W RobertsAbstract:By using DNA heteroduplexes that inhibit rewinding of the upstream part of the Transcription Bubble, we show that transcript release in termination by the enzymes Mfd and Rho is facilitated by reannealing of DNA in the upstream region of the Transcription Bubble, as is also true for termination by intrinsic terminators. We also show that, like Mfd, the Rho termination factor promotes forward translocation of RNA polymerase. These results support termination models in which external forces imposed on nucleic acids induce concerted rewinding of DNA and unwinding of the DNA/RNA hybrid, possibly accompanied by forward translocation of RNA polymerase, leading to Transcription complex dissociation.
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Role of the non-template strand of the elongation Bubble in intrinsic Transcription termination.
Journal of molecular biology, 2003Co-Authors: Andrew M. Ryder, Jeffrey W RobertsAbstract:Intrinsic Transcription terminators of Escherichia coli and other bacteria, consisting primarily of an RNA hairpin preceding a terminal uridine-rich RNA segment, suffice to dissociate the otherwise stable elongation complex of core RNA polymerase. The essential functions of the hairpin and U-rich segments have been established, although the precise mechanism of termination is unknown. We identify another element of the terminator, namely the non-template DNA strand in the region of the terminal Transcription Bubble. Failure of the terminal Bubble to rewind through complementary base-pairing strongly reduces the efficiency of terminator function, suggesting that the natural pathway of termination consists of coupled rewinding of the DNA template and unwinding of the RNA/DNA hybrid at the site of release.
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Function of a nontranscribed DNA strand site in Transcription elongation
Cell, 1994Co-Authors: Brian Z. Ring, Jeffrey W RobertsAbstract:Abstract A prolonged pause in Transcription elongation at positions +16 and +17 of the phage λ late gene operon has an important role in the modification of RNA polymerase by the λ gene Q Transcription antiterminator. Mutations included in the Transcription Bubble of the paused Transcription complex, particularly at +2 and +6, abolish pausing and the ability of Q protein to modify RNA polymerase. By transcribing heteroduplex templates made in vitro, we show that the sites identified by these mutations act through the nontranscribed strand of DNA. This result suggests unexpected regulatory functions of the nontranscribed DNA strand in Transcription.
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Structure of Transcription elongation complexes in vivo.
Science (New York N.Y.), 1992Co-Authors: Mark Kainz, Jeffrey W RobertsAbstract:The opening of duplex DNA in the elongation phase of Transcription by Escherichia coli RNA polymerase in vivo was detected at a regulatory site where a prolonged pause in Transcription occurs. Single-stranded DNA in the Transcription Bubble was identified by its reactivity with potassium permanganate (KMnO4). The elongation structure in vivo was similar to that of Transcription complexes made in vitro with some differences. The observed reactivity to KMnO4 of the DNA template strand was consistent with the existence of an RNA-DNA hybrid of about 12 nucleotides.
