Download or read book Mechanisms of sigma 54 Bacterial Transcription Activation written by Alexander Rigel Siegel and published by . This book was released on 2016 with total page 135 pages. Available in PDF, EPUB and Kindle. Book excerpt: This dissertation addresses the mechanism of [sigma]54 activation by the AAA+ ATPase transcriptional activators. The first chapter provides a general introduction to [sigma]54-mediated bacterial transcription initiation by outlining the existing structural and biochemical data on [sigma]54 and its activators. The majority of our structural information comes from high resolution structures of individual [sigma]54 and transcriptional activator domains, with the rest coming from low resolution structures that determine relative positions of the domains. The structural and biochemical properties of these domains, viewed in the context of the mechanisms of related motor proteins, led to our hypothesis of [sigma]54 activation. In this dissertation, I propose that the transcriptional activator's hexameric ATPase domain uses conserved loops, known to contact [sigma]54, to pull on and thread the [sigma]54 N-terminal activator interacting domain through its central pore. In this model, through processive rounds of ATP hydrolysis, the transcriptional activator applies enough force to generate a conformational change in the [sigma]54-RNA polymerase holoenzyme that allows it to melt DNA thus initiating transcription. The primary goal of my work has been the study of the activation mechanism using a variety of techniques, including traditional NMR-based structure determination, in vivo and in vitro biochemistry, and single molecule optical tweezers experiments. The second chapter outlines the central work of this dissertation, the structural characterization of the region of [sigma]54 responsible for contacting the activator and necessary for initiating transcription. We show that the activator interacting domain (AID) is intrinsically disordered, but becomes ordered when bound to the transcriptional activator, NtrC1, in its ATP state. In particular, we show that two predicted helices in the sequence are sufficient for activator binding with native-like affinity, and that the first helix in particular represents the primary region of contact. We applied TROSY-based NMR techniques to the structure determination of this high molecular weight complex, but most signals were broad due to dynamics or disorder, preventing a high resolution structure determination of the AID bound to the activator. This indicates that the AID does not bind the activator in a single conformation, but rather in multiple conformations each experiencing a different chemical environment. This may reflect an activation mechanism involving dynamic changes to the [sigma]54-activator interaction site. The third chapter takes an in vivo biochemical approach to study the [sigma]54 activation mechanism by characterizing the functionality of [sigma]54 with insertions and deletions near the activator interacting domain. These insertions distinguish between two competing mechanisms: whether the [sigma]54 AID only binds the surface of the activator or, as we propose, is threaded through its central pore. We find that a flexible linker region between the sites of activator and RNA polymerase binding is essential for fully functional [sigma]54. While the length of the linker does not matter, the insertion of a small, stably folded domain between these two interaction sites is detrimental to [sigma]54 activity in vivo. This is consistent with the threading of the AID and linker by the transcriptional activator, which would not be inhibited by the addition of extra unstructured residues but would be stalled by the addition of a folded domain in the linker. The fourth chapter outlines single molecule optical tweezers experiments to characterize the effect of force on a domain of [sigma]54. In particular, we examine the possibility that the [sigma]54 core binding domain (CBD), responsible for contacting core RNA polymerase, acts as a conformational fracture point that undergoes rearrangements when force is applied. The NMR structure of the CBD revealed two folded subdomains with a hydrophobic interface that could plausibly be disrupted by the force of the activator threading the N-terminus. By applying force with optical tweezers to either end of the CBD alone, we observed a separate unfolding event likely corresponding to unfolding of its seventh helix, but there was no evidence that the less stable of the two CBD subdomains unfolds before the other. Future experiments to conclusively test the force-dependent activation of [sigma]54 may require reconstituting and pulling on the full RNA polymerase holoenzyme complex, including core RNAP, promoter DNA, and full length [sigma]54. The fifth chapter steps back from the study of the [sigma]54 activation by the transcriptional activators to examine the regulation of the transcriptional activators themselves. Existing evidence shows that phosphorylation of the regulatory receiver domain of the transcriptional activator NtrC changes the population of its active and inactive states, with most states resembling the active conformation after phosphorylation. The activated receiver domain promotes the oligomerization of the ATPase domain, which in turn can activate [sigma]54. In this chapter, I studied an NtrC receiver domain from the piezophilic bacteria, Shewanella violacea, which turns on [sigma]54-dependent gene expression in response to high pressure. We hypothesized that pressure alone might drive the conformational change normally associated with phosphorylation, thereby activating S.v. NtrC independent of its normal, two-component signaling pathway. Using high pressure NMR spectroscopy, we showed that increased pressure alone does not increase the population of the S.v. NtrC receiver domain's active state. Future work must consider the pressure sensing behavior of the other components in the NtrC signaling pathway.
Download or read book Role of Escherichia Coli Sigma 54 in Binding Core RNA Polymerase and Directing Transcription Initiation written by Yin Tintut and published by . This book was released on 1995 with total page 288 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Mechanism of Stimulation of Sigma 54 and Sigma 70 RNA Polymerase Holoenzymes by Nucleotides written by Chih Min Lew and published by . This book was released on 2004 with total page 256 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Mechanism of Promoter Recognition and Transcription Regulation by the Sigma S Subunit of Escherichia Coli RNA Polymerase written by and published by . This book was released on 2004 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Diagnostic Molecular Biology written by Chang-Hui Shen and published by Elsevier. This book was released on 2023-06-29 with total page 590 pages. Available in PDF, EPUB and Kindle. Book excerpt: Diagnostic Molecular Biology, Second Edition describes the fundamentals of molecular biology in a clear, concise manner with each technique explained within its conceptual framework and current applications of clinical laboratory techniques comprehensively covered. This targeted approach covers the principles of molecular biology, including basic knowledge of nucleic acids, proteins and chromosomes; the basic techniques and instrumentations commonly used in the field of molecular biology, including detailed procedures and explanations; and the applications of the principles and techniques currently employed in the clinical laboratory. Topics such as whole exome sequencing, whole genome sequencing, RNA-seq, and ChIP-seq round out the discussion. Fully updated, this new edition adds recent advances in the detection of respiratory virus infections in humans, like influenza, RSV, hAdV, hRV but also corona. This book expands the discussion on NGS application and its role in future precision medicine. - Provides explanations on how techniques are used to diagnosis at the molecular level - Explains how to use information technology to communicate and assess results in the lab - Enhances our understanding of fundamental molecular biology and places techniques in context - Places protocols into context with practical applications - Includes extra chapters on respiratory viruses (Corona)
Download or read book Sigma 70 dependent RNA Polymerase Promoter proximal Pausing in Escherichia Coli and Its Implications for Pause mediated Transcription Regulation written by Asma Issam Hatoum and published by . This book was released on 2007 with total page 302 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Signaling Properties of the Sigma E Activation Pathway written by Irina L. Grigorova and published by . This book was released on 2005 with total page 408 pages. Available in PDF, EPUB and Kindle. Book excerpt: In a separate project, I measured the levels of RNA polymerase, sigma 70, sigmaE and sigma32 in E. coli. I use these values in an equilibrium model that considers RNA polymerase binding to DNA to explore theoretically two aspects of general regulation of transcription: recruitment of RNA polymerase to promoters by activators and competition between sigma's.
Download or read book Regulation of Transcription of the Gene for the Sigma Subunit of E Coli RNA Polymerase by a Heat Shock Promoter written by Wayne Emery Taylor and published by . This book was released on 1983 with total page 450 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Nuclear Magnetic Resonance of Biological Macromolecules Part A written by Thomas L. James and published by Academic Press. This book was released on 2001 with total page 528 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume and its companion, Volume 339, supplement Volumes 176, 177, 239, and 261. Chapters are written with a "hands-on" perspective. That is, practical applications with critical evaluations of methodologies and experimental considerations needed to design, execute, and interpret NMR experiments pertinent to biological molecules.
Download or read book An Analysis of the sigma core Interface in E Coli written by Meghan M. Sharp and published by . This book was released on 2001 with total page 326 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Mechanism of Action of the Sigma Subunit of Escherichia Coli RNA Polymerase written by Ulla Margrete Hansen and published by . This book was released on 1980 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Cryo EM Part A Sample Preparation and Data Collection written by and published by Academic Press. This book was released on 2010-09-30 with total page 466 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cryo-EM Part A: Sample Preparation and Data Collection is dedicated to a description of the instruments, samples, protocols, and analyses that belong to cryo-EM. It emphasizes the relatedness of the ideas, instrumentation, and methods underlying all cryo-EM approaches, which allow practitioners to easily move between them. Within each section, the articles are ordered according to the most common symmetry of the sample to which their methods are applied. - Includes time-tested core methods and new innovations applicable to any researcher - Methods included are useful to both established researchers and newcomers to the field - Relevant background and reference information given for procedures can be used as a guide
Download or read book Molecular Interactions Between the Transcription Factor Crl and Sigma S RNA Polymerase Holoenzyme in Escherichia Coli written by and published by . This book was released on 2013 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book Bacterial Stress Responses written by Gisela Storz and published by American Society for Microbiology Press. This book was released on 2010-11-16 with total page 1167 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gain new insight on utilizing bacterial stress responses to better combat bacterial infection with antibiotics and improve biotechnology. • Reviews the vast number of new findings that have greatly advanced the understanding of bacterial stress responses in the past 10 years. • Explores general regulatory principles, including the latest findings from genomics studies, including new research findings on both specific and general stress responses. • Details how stress responses affect the interactions between bacteria and host cells and covers bacterial stress responses in different niches and communities, with an emphasis on extreme environments.
Download or read book Bacteriophage T4 written by Christopher K. Mathews and published by . This book was released on 1983 with total page 432 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Atomic Force Microscopy of Transcription Initiation Complexes Formed by Escherichia Coli RNA Polymerase written by Sebastian Maurer and published by . This book was released on 2007 with total page 158 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book Prokaryotic Metabolism and Physiology written by Byung Hong Kim and published by Cambridge University Press. This book was released on 2019-05-16 with total page 509 pages. Available in PDF, EPUB and Kindle. Book excerpt: Extensive and up-to-date review of key metabolic processes in bacteria and archaea and how metabolism is regulated under various conditions.
