Investigation of the Topological Interactions Between Escherichia Coli Transcripton Factor Rho and RNA PDF Download
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Author: Brandt R. Burgess
Publisher:
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Category :
Languages : en
Pages : 266
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Author: Brandt R. Burgess
Publisher:
ISBN:
Category :
Languages : en
Pages : 266
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Author: Yan Wang
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Category : Adenosine triphosphatase
Languages : en
Pages : 404
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Author: James Anthony McSwiggen
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Category : Escherichia coli
Languages : en
Pages : 348
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Author: Ronnie R. Wei
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Category :
Languages : en
Pages : 296
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Author:
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ISBN:
Category :
Languages : en
Pages : 375
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Rho is a homohexameric ring-shaped protein that translocates nascent RNA through its central cleft in an ATP-dependent manner, then dissociates RNA polymerase (RNAP) from RNA and template DNA. Despite decades of study, little was known about Rho association with transcription elongation complexes (ECs); sites of Rho termination across the Escherichia coli chromosome; and the effects of the elongation factors NusG, NusA, and the nucleoid structuring protein H-NS on Rho termination. I found that Rho and RNAP have very similar distributions on DNA, suggesting that Rho associates with ECs early and throughout the process of transcription elongation. This association allows Rho to quickly respond to termination signals such as those that are unmasked when transcription and translation become uncoupled. Prior to my studies, the sites of Rho termination in E. coli were mostly unknown. I identified Rho-dependent terminators by examining the distribution of RNAP on DNA in Rho-inhibited cells. I found that Rho terminates highly structured RNAs, such as transfer RNAs (tRNAs) and small RNAs (sRNAs). This finding was surprising, because it was thought that Rho could not associate with structured RNAs. I also found that Rho terminated a small set of novel antisense transcripts that occurred within genes. Upon examining the transcriptome of cell in which Rho was inhibited, I determined that widespread increases in antisense transcription occurred throughout the genome. This finding established that a major function of Rho in vivo is to prevent elongation of antisense transcripts. Finally, I investigated the effects of NusG, NusA, and H-NS on Rho termination. I found that NusG enhances termination at a minority subset of Rho-dependent terminators that are defined by a low C/G ratio at the termination site. In contrast, a large deletion within nusA had no effect on Rho termination in vivo. I also identified a strong overlap between Rho-dependent terminators and H-NS binding sites, as well as genetic interactions between hns and rho. This led me to propose a model in which H-NS enhances Rho termination by slowing RNAP elongation, providing a longer kinetic window for Rho to terminate transcription.
Author: Erik Dean Jessen
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Category :
Languages : en
Pages : 185
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Bacterial transcription, once thought to be organized into discrete, largely non-overlapping units, has been revealed by deep cDNA sequencing to generate ubiquitous, overlapping, sense and antisense RNAs, many of which are noncoding. The function of the extensive antisense transcription of bacterial genes is controversial, and unclear in lieu of mechanistic dissection. We report here characterization of a model antisense transcription unit (bglAS) in the cryptic, H-NS-silenced b-glucoside utilization operon of E. coli K-12 (bglGFBH). bglAS was discovered because inhibition of Rho greatly increased its level and length. We created bglAS- alleles with little or no effect on bglF encoded in the sense strand by disabling the bglAS promoter with base substitutions. Although bglAS exists in a small H-NS-free island in the otherwise H-NS-coated bgl operon, the H-NS distribution is unchanged in bglAS- strains. However, when bglGFBH sense transcription was activated, bglAS decreased b-glucoside-induction of bgl gene expression via BglG-mediated antitermination of the bgl operon attenuators. Using a time series of ChIP-chip, we found that RNA polymerase progression through bglGFBH is hindered by bglAS transcription. In contrast, overexpression of bglAS RNA in trans had no effect on bgl operon induction, supporting bglAS function by transcriptional interference with bglGFBH expression. The bglAS promoter was upregulated by nitrite, consistent with bioinformatic detection of adjacent NarL/P binding sites and suggesting potential bglAS function as an environmental modulator. The proximity of bglAS to the surrounding H-NS filaments led us to investigate the interactions between transcription and H-NS. Consistent with the inability of transcription to affect H-NS binding patterns at the bgl operon, the genome-scale H-NS distribution exhibited minimal change when all transcription was inhibited with rifampicin. However, deletion of H-NS and StpA, an H-NS paralog, resulted in increased progression of RNAP along active, H-NS bound transcription units. Analyzing NET-seq data suggested that H-NS inhibits RNAP progression by promoting pausing of RNAP at non-canonical pause sequences. In contrast to H-NS, the nucleoid-associated protein HU was found to have a distribution pattern mirroring highly active transcription units. Upon further investigation, HU most closely correlated with the presence of R-loops, stable RNA:DNA hybrids external of RNAP. R-loop levels are independent of the presence of HU, suggesting HU is not involved in the resolution of R-loops. Instead, deletion of HU confers sensitivity to reactive oxygen species, and we propose a model by where HU binds to the areas around R-loops to protect DNA from mutagenesis. The combined studies presented here are a significant advancement in our understanding of the function of antisense transcripts and the interaction between transcription and nucleoid-associated proteins.
Author: Michael John Allen Bellecourt
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Category :
Languages : en
Pages : 0
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Cellular organisms use RNA polymerases (RNAPs) to express genes through the process of transcription. Accurate and timely RNA synthesis requires regulation of all steps of a complex transcription cycle, a regulatory framework involving the careful interplay of interactions between RNAP, the sequences and structure of template DNA and the nascent RNA, and myriad transcription factors that associate with both RNAP and the chromosome. At the end of a transcription unit, after kilobases of transcription, the elongation complex must efficiently release RNA and RNAP from DNA over a 2-3 nucleotide window. In bacteria, there are two programmed termination mechanisms. Intrinsic terminators utilize a GC-rich dyad immediately upstream to a U-rich tract that, together, promotes termination in the absence of additional transcription factors. Rho-dependent termination utilizes the RNA helicase Rho, which binds unstructured RNAs and utilizes its motor activity to extract the nascent RNA. Prior to my studies, the role of nucleic-acid sequence and structure in these mechanisms were well-characterized, but the role of protein conformation changes were largely unknown. To investigate how movements by the RNAP clamp affected intrinsic termination, I disentangled the steps of elongation, commitment, and dissociation, which are conflated in traditional termination measures. I found that clamp movements affected termination efficiency due to effects on elongation, not termination itself. Next, I found that clamp opening is necessary for release of RNAP from DNA, but not release of RNA. I also demonstrated that restricting movements of the trigger loop prior to commitment inhibits intrinsic termination, in agreement with a recently proposed multistate-multipath model of intrinsic termination. My studies also determined how the transcription factor NusG promotes Rho-dependent termination. After binding C-rich RNAs in its secondary site, the Rho ring makes small amino acid rearrangements in its C-terminal domain to induce ring closure. At subpar RNAs that Rho cannot effectively bind, NusG associates near this amino acid switch network, inducing the necessary rearrangements. My studies underscore the importance of protein movements during the termination process, from small-scale Brownian motion to large-scale movements of protein modules, and further demonstrate that RNAP might remain associated with DNA after termination.
Author: Marcel Geertz
Publisher:
ISBN:
Category :
Languages : en
Pages : 172
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For understanding the concerted molecular interactions aiding into the assembly of cellular transcription programs it is essential to reveal a general mechanism which directs the transcription machinery to particular sets of gene promoters in a spatially and temporally ordered way. A global regulatory network involving nucleoid-associated proteins (NAPs), DNA topoisomerases and the components of RNA polymerase (RNAP) is implicated in this mechanism. While DNA supercoiling is a global regulator of gene transcription, in Escherichia coli the reorganisation of cellular transcription is associated with concerted alterations of two principal parameters: the composition of RNAP and the structural organisation of the nucleoid DNA, both of which change with growth. The mechanism of coupling between compositional alterations of NAPs, RNAP and topological changes of DNA remains unclear. Different facets of transcriptional regulation were studied to reveal this coupling mechanism. This work shows a determinative role of RNAP composition for the overall supercoiling level and reveals structural coupling between RNAP and the NAPs as a mechanism coordinating global transcription. This work further shows that the NAPs regulate the genomic distributions of DNA supercoils, whereas the sensing and utilization of the supercoils is in turn dependent on the composition of RNAP. For one of the NAPs, H-NS, the binding site consensus has been identified and the genomic transcript profiles were investigated to elucidate, how the genomic distributions of these sites can regulate supercoil dynamics. This work distinguishes the transcriptional regulatory network of functionally related genes from the network of spatially neighboring genes and shows their tight interdependence in organizing global transcription. In summary, this work provides novel insights in the organizational principles and understanding of molecular mechanisms underlying the coordination of bacterial gene expression.
Author:
Publisher:
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Category :
Languages : en
Pages : 586
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Bacteria must change their cellular composition in response to changing environmental conditions and stress. They implement these changes by altering gene expression and/or protein activity. Central to changes in gene expression during stress responses is regulation of transcription. In bacteria, transcription is directed by a suite of RNA polymerase (RNAP) holoenzymes, comprised of a core enzyme, possessing the catalytic activity for RNA synthesis, and one of a collection of transcription initiation factors, known as Ï3 factors, which recognizes promoter DNA sequences upstream of genes. Increasing the formation of a particular type of RNAP holoenzyme by switching the Ï3 factor will lead to expression of its cognate regulon. My thesis research focuses on deciphering the complex molecular interactions between the transcription factor Crl and the general stress response sigma factor, Sigma S, in order to understand Escherichia coli's response to stress at the molecular level. Crl is a small (133 amino acids in E. coli) protein that stimulates transcription mediated by Sigma S. At the outset of my research, little was known about how Crl interacts with the transcription machinery to stimulate Sigma S-mediated transcription. I used a combination of biochemical, genetic, and molecular biological techniques in vitro and in vivo to identify key interactions and gain insight into the mechanism of transcriptional regulation by Crl. I identify a key binding determinant (the DPE motif) in Sigma S conserved domain 2 underlying its specific recognition by Crl. I demonstrate directly that Crl requires this determinant for positive regulation of Sigma S-mediated transcription and to enhance Sigma S holoenzyme formation. I made the primary Sigma factor recognizable by Crl by substitution of the DPE motif along with deletion of a large non-conserved region (NCR). I also localized the area of Crl required for the interaction with Sigma S to Crl's conserved central cleft. Finally, I identified an interaction between the Beta prime subunit of core RNAP and Crl, which occurs only in the context of the holoenzyme, that has begun to clarify the mechanism by which Crl functions as a Sigma S-specific RNAP holoenzyme assembly factor.
Author: Sebastian Maurer
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ISBN:
Category :
Languages : en
Pages : 158
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As much as protein - DNA interactions depend on the conformation of the binding protein they are also influenced by the local geometry of the DNA. Since the topology of DNA is continuously changing due to ongoing replication, transcription and associated fluctuations of topoisomerase activity, the accessibility and configuration of the recognition elements for proteins, the major grooves for example, are also subject to changes. Thus many protein-DNA interactions are affected by a change of the superhelicity of DNA, although to different extent. The work at hand focuses on the effect of DNA topology on regulation of gene transcription - a relationship studied on the example of two supercoiling sensitve E.Coli promoters: The fis (factor of inversion stimulation) promoter and the Tyrosine tRNA promoter. By utilizing Atomic Force Microscopy (AFM) this study demonstrates both: a complex of two RNAP molecules forming a dimer on binding to the divergent promoter module consisting of fisP1 and fisPdiv sites, as well as the structures of FIS and RNAP bound to the tyrT promoter either separately or together - thereby visualizing a ó70-RNAP containing transcription initiation complex for the first time. The models for the transcriptional activation of the fis and the tyrT promoters presented in this work were proposed under consideration of the nucleoprotein complex structures visualized by AFM and supported by further biochemical data. Despite the differences in sequence organisation of the fis and tyrT promoters, both models share a common feature: A transcription activation mechanism which acts through the optimization of the local superhelical density.
