Download or read book Single molecule Fluorescence Analysis of Opening and Closing of the RNA Polymerase Clamp written by Anirban Chakraborty and published by . This book was released on 2013 with total page 111 pages. Available in PDF, EPUB and Kindle. Book excerpt: Crystal structures of RNA polymerase (RNAP) indicate that the RNAP [beta]' pincer ("clamp") can exist in conformational states, ranging from a fully open conformation that permits entry and exit of DNA, to a fully closed conformation that prevents entry and exit of DNA. It has been hypothesized that the clamp also adopts multiple conformational states in solution and conformational changes in the clamp are important for function. In this work, a single-molecule fluorescence resonance energy transfer (smFRET) approach was developed that enables determination of RNAPclamp conformation in solution. smFRET was measured between a probe at the tip of the RNAP clamp and a probe at a fixed reference point in RNAP. A computational framework was then employed to interpret measured FRET efficiencies in terms of structural changes. Using this approach, RNAP clamp conformation was defined in each step of?70-dependent transcription initiation and elongation and in each step in?54-dependent transcription initiation. Additionally, effects of four RNAP inhibitors, myxopyronin, corallopyronin, ripostatin and Gp2 on RNAP clamp conformation were assessed. It was observed that the clamp is predominantly open in free RNAP and in all steps leading up to the formation of a catalytically-competent-transcription-initiation complex. Upon formation of a catalytically-competent-transcription-initiation complex, the clamp closes, and continues to remain closed during transcription elongation. It was further observed that myxopyronin, corallopyronin, ripostatin and Gp2, prevent opening of the RNAP clamp. The results lead to the proposal that, the open clamp state is important for entry of DNA into, and unwinding of DNA in, the RNAP active center cleft during formation of a catalytically-competent-transcription initiation complex. The results lead to the proposal that, after entry of DNA into the RNAP active-center cleft upon formation of the catalytically competent transcription initiation complex, electrostatic interactions between the negatively charged DNA and the positively charged inner facet of the clamp, induce and/or stabilize clamp closure. The results are in agreement with the proposal that, clamp closure is important for stability of the catalytically competent transcription initiation complex and for stability and processivity of the transcription elongation complex.

Download or read book Single Molecule Tools Part A Fluorescence Based Approaches written by and published by Academic Press. This book was released on 2010-08-17 with total page 559 pages. Available in PDF, EPUB and Kindle. Book excerpt: Single molecule tools have begun to revolutionize the molecular sciences, from biophysics to chemistry to cell biology. They hold the promise to be able to directly observe previously unseen molecular heterogeneities, quantitatively dissect complex reaction kinetics, ultimately miniaturize enzyme assays, image components of spatially distributed samples, probe the mechanical properties of single molecules in their native environment, and "just look at the thing" as anticipated by the visionary Richard Feynman already half a century ago. Single Molecule Tools, Part A: Fluorescence Based Approaches captures a snapshot of this vibrant, rapidly expanding field, presenting articles from pioneers in the field intended to guide both the newcomer and the expert through the intricacies of getting single molecule tools. - Includes time-tested core methods and new innovations applicable to any researcher employing single molecule tools - 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 to developing protocols in a number of disciplines

Download or read book Ensemble Fluorescence Resonance Energy Transfer Analysis of RNA Polymerase Clamp Conformation written by Dongye Wang and published by . This book was released on 2008 with total page 143 pages. Available in PDF, EPUB and Kindle. Book excerpt: Crystal structures of RNA Polymerase (RNAP) and RNAP complexes indicate that the RNAP beta' pincer ("clamp") can exist in a range of conformational states, ranging from a fully open conformation that permits entry and exit of DNA, to a fully closed conformation that prevents entry and exit of DNA. Clamp closure involves a swinging motion of the beta' pincer by the switch region at the base of the beta' pincer. In order to define RNAP clamp conformation in solution, we have used fluorescence resonance energy transfer (FRET) to monitor the distance between a first fluorescent probe, serving as donor, incorporated at the tip of the beta' pincer and a second fluorescent probe, serving as acceptor, incorporated at the tip of the beta' pincer. We have developed a procedure that permits incorporation of a fluorescent probe within a protein. The procedure involves preparation of a protein containing the azide-containing unnatural amino acid p-azidophenylalanine at the site of interest, followed by incorporation of a fluorescent probe through azide-specific chemical modification (accomplished by Staudinger-Bertozzi ligation using a phosphine derivative of the fluorescent probe). We have used this procedure to incorporate fluorescent probes at the tips of the RNAP beta' pincer and RNAP beta pincer. We have used the resulting labeled RNAP derivatives in FRET experiments addressing opening and closing of the RNAP active-center-cleft in transcription initiation and elongation and in FRET experiments addressing effects of small-molecule effectors, myxopyronin (Myx), corallopyronin (Cor), ripostatin (Rip), and lipiarmycin (Lpm), on opening and closing of the RNAP active-center cleft. Results indicate that: (1) RNAP holoenzyme in solution exists predominantly in a partly closed clamp conformational state (2) the RNAP clamp closes upon formation of the RNAP-promoter open complex, yielding a fully closed clamp conformational state (3) the RNAP clamp remains closed--and exhibits no further change in mean clamp conformation--upon formation of RNAP-promoter initial transcribing complexes and transcription elongation complexes. The results support the proposal that Myx, Cor, Rip and Lpm bind to an RNAP- switch-region conformational state and Myx. Cor, Rip and Lpm inhibit RNAP function by trapping the RNAP switch region in this conformational state, thereby interfering with conformational cycling of RNAP clamp important for RNAP function.

Download or read book Displacement of DNA molecular mimic R1 1 from the RNA Polymerase Active center Cleft written by Qumiao Xu and published by . This book was released on 2013 with total page 102 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Bacterial Transcriptional Control written by Irina Artsimovitch and published by Humana. This book was released on 2016-10-05 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Single Molecule Fluorescence Studies of the RNA Polymerase II Elongation Complex written by Joanna Andrecka and published by . This book was released on 2009 with total page 112 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book High resolution Single molecule Measurements of Transcription and RNA Folding written by William James Greenleaf and published by . This book was released on 2007 with total page 296 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Determining the Rate of Transcription of T7 RNA Polymerase Using Single Molecule Fluorescence Imaging written by Dawn Renee Nicholas and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Handbook of Single Molecule Biophysics written by Peter Hinterdorfer and published by Springer Science & Business Media. This book was released on 2009-12-24 with total page 634 pages. Available in PDF, EPUB and Kindle. Book excerpt: This handbook describes experimental techniques to monitor and manipulate individual biomolecules, including fluorescence detection, atomic force microscopy, and optical and magnetic trapping. It includes single-molecule studies of physical properties of biomolecules such as folding, polymer physics of protein and DNA, enzymology and biochemistry, single molecules in the membrane, and single-molecule techniques in living cells.

Download or read book Single Molecule Study of RNA Polymerase written by Keir Cajal Neuman and published by . This book was released on 2002 with total page 212 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Protein Nucleic Acid Interactions written by Phoebe A. Rice and published by Royal Society of Chemistry. This book was released on 2008-05-22 with total page 417 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides both in-depth background and up-to-date information in this area. The chapters are organized by general themes and principles, written by experts who illustrate topics with current findings. Topics covered include: - the role of ions and hydration in protein-nucleic acid interactions - transcription factors and combinatorial specificity - indirect readout of DNA sequence - single-stranded nucleic acid binding proteins - nucleic acid junctions and proteins, - RNA protein recognition - recognition of DNA damage. It will be a key reference for both advanced students and established scientists wishing to broaden their horizons.

Download or read book Sequence Dependent Pausing by RNA Polymerase written by Kristina Marie Herbert and published by . This book was released on 2007 with total page 205 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Single Molecule Analysis written by Iddo Heller and published by Springer Nature. This book was released on 2023-11-13 with total page 511 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Probing the Conformational Distribution of the T7 RNA Polymerase Promoter DNA Sequence Using Single Molecule Fluorescene Spectroscopy written by Gerard J. Hilinski and published by . This book was released on 2004 with total page 254 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Promoter Melting by RNA Polymerase written by Andrey Revyakin and published by . This book was released on 2003 with total page 510 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Chemical Biology of Nucleic Acids written by Volker A. Erdmann and published by Springer Science & Business. This book was released on 2014-04-22 with total page 599 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume contains 29 engrossing chapters contributed by worldwide, leading research groups in the field of chemical biology. Topics include pre-biology; the establishment of the genetic code; isomerization of RNA; damage of nucleobases in RNA; the dynamic structure of nucleic acids and their analogs in DNA replication, extra- and intra-cellular transport; molecular crowding by the use of ionic liquids; new technologies enabling the modification of gene expression via editing of therapeutic genes; the use of riboswitches; the modification of mRNA cap regions; new approaches to detect appropriately modified RNAs with EPR spectroscopy and the use of parallel and high-throughput techniques for the analysis of the structure and new functions of nucleic acids. This volume discusses how chemistry can add new frontiers to the field of nucleic acids in molecular medicine, biotechnology and nanotechnology and is not only an invaluable source of information to chemists, biochemists and life scientists but will also stimulate future research.

Download or read book Single Molecule Enzymology Nanomechanical Manipulation and Hybrid Methods written by and published by Academic Press. This book was released on 2017-01-03 with total page 486 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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