Single Molecule Studies of Bacterial RNA Polymerase Dynamics PDF Download
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Author: Timothy T. Harden
Publisher:
ISBN:
Category : Messenger RNA.
Languages : en
Pages : 107
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Book Description
Author: Timothy T. Harden
Publisher:
ISBN:
Category : Messenger RNA.
Languages : en
Pages : 107
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Book Description
Author: Yazan Khalaf Alhadid
Publisher:
ISBN:
Category :
Languages : en
Pages : 146
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Book Description
Transcription of genomic DNA of all organisms is carried out by members of the multi-subunit RNA polymerase family. Regulation of RNA polymerase localization and activity underlies cellular homeostasis, division, and response to environmental cues. The catalytic mechanism, overall architecture, and many sequence and structural features of bacterial RNA polymerase are conserved in its Archaeal and Eukaryotic counterparts. The human RNA polymerase II (Pol II) is responsible for transcription of all protein-coding and many non-coding genes. The majority of current knowledge on RNA polymerases and their mechanism at different steps in transcription derives from extensive work done using classical biochemical, genetic and structural biology methods. However, the use of single-molecule approaches addressed crucial questions on the function and mechanism of RNA polymerases during transcription, which were not possible to answer with ensemble-based approaches due to averaging effects. A useful fluorescence-based single-molecule technique to measure distances on the molecular scale and monitor dynamics is F rster resonance energy transfer (FRET). Here, I report on the development of diffusion-based single-molecule FRET (smFRET) methods to investigate different steps in transcription by the in vitro reconstituted human Pol II system. Using an assay that monitors the FRET changes between fluorescent dyes in the unwound region of promoter DNA (transcription bubble), I demonstrated the effect of certain components of the reconstituted system on the relative size of the transcription bubble. I also detail the optimizations done to enhance the affinity of single-stranded DNA (ssDNA) FRET probes to complementary target sequences. These ssDNA FRET probes were used to investigate the effect of certain components of the reconstituted system on Pol II activity by measuring the relative levels of RNA product. In addition to studies on the Pol II system, I report on the effect of the 5'-group of nascent RNA on the stability of the Escherichia coli RNA polymerase (RNAP) transcription bubble. I show how the presence of a 5'-monophosphate appears to destabilize the open bubble while a 5'-hydroxyl has no effect. Finally, I describe the work done on a project I took part in that identified a previously uncharacterized RNAP paused complex in initiation. We demonstrate that RNAP complexes undergoing initial transcription can enter the inactive paused state by backtracking. I also demonstrate how the presence of a 5'-triphosphate rapidly enhances entrance of RNAP complexes undergoing initial transcription into an inactive paused complex.
Author: Henri C. Buc
Publisher: Royal Society of Chemistry
ISBN: 0854041346
Category : Science
Languages : en
Pages : 353
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Book Description
This book, written by expert scientists in the field, analyses how these diverse fields of research interact on a specific example - RNA polymerase. The book concentrates on RNA polymerases because they play a central role among all the other machines operating in the cell and are the target of a wide range of regulatory mechanisms. They have also been the subject of spectacular advances in their structural understanding in recent years, as testified by the attribution of the Nobel prize in chemistry in 2006 to Roger Kornberg. The book focuses on two aspects of the transcription cycle that have been more intensively studied thanks to this increased scientific cooperation - the recognition of the promoter by the enzyme, and the achievement of consecutive translocation steps during elongation of the RNA product. Each of these two topics is introduced by an overview, and is then presented by worldwide experts in the field, taking the viewpoint of their specialty. The overview chapters focus on the mechanism-structure interface and the structure-machine interface while the individual chapters within each section concentrate more specifically on particular processes-kinetic analysis, single-molecule spectroscopy, and termination of transcription, amongst others. Specific attention has been paid to the newcomers in the field, with careful descriptions of new emerging techniques and the constitution of an atlas of three-dimensional pictures of the enzymes involved.
Author: Robert Landick
Publisher: Royal Society of Chemistry
ISBN: 1839160667
Category : Science
Languages : en
Pages : 295
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Book Description
To thrive, every living cell must continuously gauge and respond to changes in its environment. These changes are ultimately implemented by modulating gene expression, a process that relies on transcription by Nature’s most multivalent molecular machine, the RNA polymerase. This book covers progress made over the past decade understanding how this machine functions to compute the cellular state, from the atomistic structural level responsible for chemistry to the integrative level at which RNA polymerase interacts with the other key molecular machineries of the cell.
Author: Irina Artsimovitch
Publisher: Humana
ISBN: 9781493954674
Category : Science
Languages : en
Pages : 0
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Book Description
This volume is designed to be a resource of proven techniques and approaches for probing the activities of bacterial, eukaryotic, and archaeal RNA polymerases. This book features a collection of in vitro and in vivo technologies that will permit researchers to purify and probe the position and stability of RNA polymerase complexes at different points of the transcription cycle, analyze the various translocations and intermolecular movements associated with catalysis, define recruitment strategies, probe the roles of transcription factors in each stage of the cycle, highlight conserved and disparate fidelity mechanisms, analyze the resultant transcripts, and study coordination of the nascent mRNA synthesis by the RNA polymerase and mRNA translation by the ribosome. Written in the highly successful Methods of Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and key tips on troubles troubleshooting and avoiding known pitfalls. Practical and timely, Bacterial Transcriptional Controls: Methods and Protocols highlights the breadth and depth of techniques that are likely to continue shaping the transcription community in the future.
Author: Alexander E. Knight
Publisher: Academic Press
ISBN: 008092123X
Category : Science
Languages : en
Pages : 369
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Book Description
Single molecule techniques, including single molecule fluorescence, optical tweezers, and scanning probe microscopy, allow for the manipulation and measurement of single biological molecules within a live cell or in culture. These approaches, amongst the most exciting tools available in biology today, offer powerful new ways to elucidate biological function, both in terms of revealing mechanisms of action on a molecular level as well as tracking the behaviour of molecules in living cells. This book provides the first complete and authoritative treatment of this rapidly emerging field, explicitly from a biological perspective.The contents are organized by biological system or molecule. Each chapter discusses insights that have been revealed about their mechanism, structure or function by single molecule techniques. Among the topics covered are enzymes, motor proteins, membrane channels, DNA, ribozymes, cytoskeletal proteins, and other key molecules of current interest. An introduction by the editor provides a concise review of key principles and an historical overview. The last section discusses applications in molecular diagnostics and drug discovery. - Organized by biological system or molecule - Each chapter discusses insights into mechanism of action, structure, and function - Covers enzymes, motor proteins, membrane channels, DNA, ribozymes, etc - Includes an introduction to key principles and an historical overview - Discusses applications in molecular diagnostics and drug discovery - Provides an expert's perspective on future development
Author: Erwin J. G. Peterman
Publisher: Humana Press
ISBN: 9781617792816
Category : Science
Languages : en
Pages : 317
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Book Description
Life scientists believe that life is driven, directed, and shaped by biomolecules working on their own or in concert. It is only in the last few decades that technological breakthroughs in sensitive fluorescence microscopy and single-molecule manipulation techniques have made it possible to observe and manipulate single biomolecules and measure their individual properties. The methodologies presented in Single Molecule Techniques: Methods and Protocols are being applied more and more to the study of biologically relevant molecules, such as DNA, DNA-binding proteins, and motor proteins, and are becoming commonplace in molecular biophysics, biochemistry, and molecular and cell biology. The aim of Single Molecule Techniques: Methods and Protocols is to provide a broad overview of single-molecule approaches applied to biomolecules on the basis of clear and concise protocols, including a solid introduction to the most widely used single-molecule techniques, such as optical tweezers, single-molecule fluorescence tools, atomic force microscopy, magnetic tweezers, and tethered particle motion. Written in the highly successful Methods in Molecular BiologyTM series format, chapters contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and accessible, Single Molecule Techniques: Methods and Protocols serves as an ideal guide to scientists of all backgrounds and provides a broad and thorough overview of the exciting and still-emerging field of single-molecule biology.
Author: Iddo Heller
Publisher: Springer Nature
ISBN: 1071633775
Category : Science
Languages : en
Pages : 511
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Book Description
This third edition volume expands on the previous editions with new discussions on the latest techniques and developments in the field. The chapters in this book are organized into four parts, and cover topics such as optical tweezers; single-molecule fluorescence tools; atomic force microscopy; magnetic tweezers; applications to virus protein shells, unfolding of proteins, nucleic acids, motor proteins, in vivo and in vitro; and protocols to establish specific surface interactions and perform force calibration. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Single Molecule Analysis: Methods and Protocols, Third Edition is a valuable resource for all researchers who want to learn more about this exciting and still expanding field. Chapters 2, 7, 8, 9, 12, 18, and 19 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Author:
Publisher: Academic Press
ISBN: 0128095032
Category : Science
Languages : en
Pages : 486
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Book Description
Single-Molecule Enzymology, Part B, the latest volume in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in single-molecule enzymology, and includes sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field - Covers research methods in single-molecule enzymology - Contains sections on such topics as force-based and hybrid approaches, fluorescence, high-throughput sm enzymology, and nanopore and tethered particle motion
Author: Joanna Andrecka
Publisher:
ISBN:
Category :
Languages : en
Pages : 112
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Book Description
