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 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 An Optical Tweezers Assay for the Study of T7 RNA Polymerase written by Gary Mark Skinner and published by . This book was released on 2001 with total page 0 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 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 Study of Transcription by RNA Polymerase I written by Ana Lisica and published by . This book was released on 2013 with total page 116 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Conformational Change of DNA Bound by T7 RNA Polymerase During Transcription Initiation written by Fiona Patricio and published by . This book was released on 2001 with total page 92 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single molecule Studies of Different Steps in Human RNA Polymerase II and Bacterial RNA Polymerase Transcription written by Yazan Khalaf Alhadid and published by . This book was released on 2018 with total page 146 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book A Novel Approach to Studying Transcription by T7 RNA Polymerase Through Fluorescence Techniques written by Robert Loree and published by . This book was released on 2002 with total page 118 pages. Available in PDF, EPUB and Kindle. Book excerpt:
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 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 Fluorescence Anisotropy to Determine a Dissociation Constant Kd for the T7 RNA Polymerase to Its Promoter DNA written by Alicia Shapiro Romero and published by . This book was released on 1995 with total page 120 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Probing DNA Conformational Change During Transcription by Bacteriophage T7 RNA Polymerase Using Fluorescence Resonance Energy Transfer written by Edward F. Chang and published by . This book was released on 1997 with total page 212 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Single molecule Measurements of Transcript Elongation and Termination by RNA Polymerase written by Matthew Herbert Larson and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Transcription by RNAP is highly regulated in both prokaryotic and eukaryotic cells, and the ability of the cell to differentiate and respond to its environment is largely due to this regulation. During elongation, for example, RNAP is known to momentarily halt in response to certain cellular signals, and this pause state has been implicated in the regulation of gene expression in both prokaryotic and eukaryotic organisms. In addition, once RNAP reaches the end of a gene, it must reliably terminate and release the newly-transcribed RNA, providing another potential point of regulation within different cell types. Both of these steps are crucial to ensure proper gene expression. In this dissertation, I focus on transcription elongation by both prokaryotic and eukaryotic RNA polymerases, as well as their regulation through pausing and termination. To probe the role of RNA hairpins in transcriptional pausing, a novel single-molecule "RNA-pulling" assay was used to block the formation of secondary structure in the nascent transcript. Force along the RNA did not significantly affect transcription elongation rates, pause frequencies, or pause lifetimes, indicating that short "ubiquitous" pauses are not a consequence of RNA hairpins. Force-based single-molecule techniques were also used to study the mechanism and energetics of transcription termination in bacteria. The data suggest two separate mechanisms for termination: one that involves hypertranslocation of RNAP along the DNA, and one that involves shearing of the RNA:DNA hybrid within the enzyme. In addition, a quantitative energetic model is presented that successfully predicts the termination efficiency of both wild-type and mutant terminators. Finally, the implementation of a novel optical-trapping assay capable of directly observing transcription by eukaryotic RNA polymerase II (RNAPII) molecules is described. This approach was used to probe the RNAPII nucleotide-addition cycle, as well as the role of the trigger loop (a conserved subdomain) in elongation. The results are consistent with a Brownian ratchet model of elongation which incorporates a secondary NTP binding site, and the trigger loop was found to modulate translocation, NTP binding, and catalysis, as well as substrate selection and mismatch recognition by RNAPII.
Download or read book Cumulated Index Medicus written by and published by . This book was released on 1999 with total page 1844 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 Modelling Natural Action Selection written by Tony J. Prescott and published by . This book was released on 2007 with total page 864 pages. Available in PDF, EPUB and Kindle. Book excerpt:
