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 Probing Single stranded DNA Structure and Conformational Transitions with Single molecule Fluorescence Spectroscopy written by Andrea H. Kurtz and published by . This book was released on 2008 with total page 442 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 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 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 Index Medicus written by and published by . This book was released on 2004 with total page 2068 pages. Available in PDF, EPUB and Kindle. Book excerpt: Vols. for 1963- include as pt. 2 of the Jan. issue: Medical subject headings.

Download or read book Probing Elongational Processivity During Transcription by T7 RNA Polymerase Using a Novel FRET based Conformational Reporting System written by Shivang Shah and published by . This book was released on 2003 with total page 170 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 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 Single Molecule Fluorescence Spectroscopy of the Folding of a Repeat Protein written by Sharona Cohen and published by Springer. This book was released on 2015-10-17 with total page 70 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this thesis single-molecule fluorescence resonance energy transfer (FRET) spectroscopy was used to study the folding of a protein that belongs to the large and important family of repeat proteins. Cohen shows that the dynamics of the expanded conformations is likely to be very fast, suggesting a spring-like motion of the whole chain. The findings shed new light on the elasticity of structure in repeat proteins, which is related to their function in binding multiple and disparate partners. This concise research summary provides useful insights for students beginning a PhD in this or a related area, and researchers entering this field.

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 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 Measurement of Molecular Conformations and Dynamics Using Single Molecule Fluorescence Techniques written by Huimin Chen and published by . This book was released on 2012 with total page 122 pages. Available in PDF, EPUB and Kindle. Book excerpt: The use of fluorescence spectroscopy to study biological problems has gained popularity over the past few decades. Beyond a spatial understanding provided by microscopy, fluorescence techniques like Fluorescence Correlation Spectroscopy (FCS) and fluorescence lifetime spectroscopy can also elucidate the important temporal dynamics of molecular conformations. We have applied FCS to study the conformational fluctuations in a model protein apomyoglobin. By pushing the technical limitations of FCS, we were able to observe conformational dynamics spanning two orders of magnitude in time (10[-]6 to 10[-]3 seconds). We found that the amplitude of fluctuations changes as the molecule becomes unfolded, with principal shifts of amplitudes and timescales occurring at the transition across the molten globule state. We also measured the diffusion of apomyoglobin, confirming theoretical predictions of less compaction of the molecule upon acid denaturation. We were able to observe the fluctuations in apomyoglobin using FCS due to the quenching of an N-terminal labeled Alexa488 fluorophore by contact with various amino acids. We showed that quenching can occur with up to four amino acids. We investigated the mechanisms of quenching using a combination of fluorescence intensity and lifetime measurements. We showed that quenching takes place through a combination of static and dynamic mechanisms. Our results demonstrate that care needs to be taken when making quan- titative measurements of fluorescently labeled proteins. Coupling single molecule functionality to fluorescence techniques allows researchers to discern subpopulations within ensembles. We used single molecule Förster Resonance Energy Transfer (smFRET) to measure the end-to-end distances in single stranded nucleic acids as a probe of the flexibility. We also measured the radius of gyration using small angle X-ray scattering, and extracted parameters of polymer properties that fit the data from both techniques. We observed clear differences between our model single stranded DNA (ssDNA) and RNA (ssRNA). We also observed a difference between the screening efficiency of monovalent and divalent cations. By characterizing the intrinsic differences in nucleic acids and its dependence on the ionic strength, we hope to improve our understanding of the mechanisms of RNA folding and the role of ions in the process.

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 Studying Topologically Complex DNA at the Single molecule Level written by Wan Yuan Beatrice Soh and published by . This book was released on 2020 with total page 222 pages. Available in PDF, EPUB and Kindle. Book excerpt: Over two decades ago, with advances in microfabrication techniques and fluorescence microscopy, single-molecule studies emerged as a powerful approach to investigate polymer dynamics at the molecular level. By providing a platform for the direct observation and precise manipulation of individual polymer molecules, single-molecule studies allow for the probing of microscopic interactions that give rise to the macroscopic properties of the polymer system. Single molecule studies have been widely used to investigate the static and dynamic properties of double-stranded deoxyribonucleic acid (DNA) as a model polymer. Such studies not only help to develop a fundamental understanding of key topics in polymer physics that cannot be easily accessed via traditional bulk experimental methods, but also facilitate the development of emerging DNA mapping and sequencing techniques. The majority of single-molecule studies to date have involved linear DNA molecules. It is known that topological constraints on the molecular level have a signicant influence on polymer dynamics. A nascent area in the field of polymer physics is the study of polymers with complex topologies. In this thesis, we present a series of single-molecule experiments and Brownian dynamics simulations used to investigate the polymer physics of topologically complex DNA. Specically, we focus on knotted polymers, ring polymers and catenated polymer networks. To investigate the impact of a knot on polymer dynamics, we employ a combined approach of single-molecule experiments and Brownian dynamics simulations. We study experimentally the steady-state behavior of knotted polymers in planar elongational fields and nd that the presence of a knot leads to a faster relaxation time and, accordingly, a shift in the coilstretch transition for the molecule. In consequence, the untying of a knot near the coilstretch transition can give rise to dramatic changes in chain conformation. We use Brownian dynamics simulations to study in detail the impact of the knot untying process on polymer dynamics in planar elongational fields and complement the simulations with experimental results. As a knot moves o the chain in an elongational field, the knot size changes due to the non-uniform tension prole along the chain and causes a change in the eective Weissenberg number, which in turn leads to a change in chain extension. With the use of simulations, we further investigate the knot untying process by probing the topological pathway of an untying knot. We study the distributions of knot conformational states and knot untying pathways on uniformly tensioned chains and chains subjected to elongational fields, and demonstrate that external fields can be used to influence how a knot unties from a chain. Next, we shift focus to ring polymers. We use single-molecule experiments to study the dynamics of self-entangled circular DNA. Our results demonstrate that ring polymers can self-entangle by forming self-threadings, and that such threadings can lead to a signicant slowdown in polymer dynamics. It seems counterintuitive that self-entanglements can arise in ring polymers, which lack chain ends. To delve into the physics of self-entanglements on circular chains, we implement a macroscopic system that allows for the direct visualization of chain conformation. We investigate the formation of self-entanglements on granular chains subjected to a tumbling motion, and use the well-studied self-entanglements on linear chains as a framework for interpreting self-entanglements on circular chains. We develop a method to characterize the self-entanglements on circular chains with known topological descriptors from knot theory and propose a general mechanism for the self-entanglement of circular chains. Finally, we consider the deformation dynamics of catenated DNA networks. A kinetoplast is a complex network of catenated DNA rings that resembles a two-dimensional polymeric system. We perform single-molecule experiments to study the deformation response of kinetoplasts in a planar elongational field. Our results demonstrate that kinetoplasts deform in a stagewise fashion and undergo transient deformation at large strains, as a result of conformational rearrangements from a metastable state. In contrast to linear polymers that display a coil-stretch transition, kinetoplasts do not exhibit an abrupt transition between the non-deformed and deformed states.

Download or read book High Resolution Measurements of RNA Polymerase with Nanopore Tweezers written by Ian C. Nova and published by . This book was released on 2020 with total page 164 pages. Available in PDF, EPUB and Kindle. Book excerpt: RNA polymerase (RNAP) is the molecular machine responsible for transcription, the process of making RNA from a DNA template. While the overall role of RNAP in this central cellular process is clear (RNAP moves along double stranded DNA, extending an RNA chain by 1 base with each step), questions remain about the timing and order of events within each reaction cycle [1]. With bulk biochemical assays that average over an ensemble of molecules, determining the dynamics of individual RNAP enzymes during transcription is difficult. For this reason, experiments that capture and observe single RNAP molecules have been particularly elucidating. Specificacally, optical tweezers experiments, in which the position of single RNAP molecules along DNA are tracked over time, have helped develop and test various kinetic models for RNAP transcription [2]. By varying applied force and nucleotide triphosphate (NTP, the substrate for transcription) concentration, these experiments probe the energy landscape and reveal the coupling between chemical hydrolysis and mechanical motion [3]. However, in physiologically relevant conditions, single steps of RNAP occur too quickly and over too small a distance (1 base pair) to be detected by optical tweezers [4]. This necessitates averaging over multiple steps in the reaction cycle, prohibiting direct observation of individual enzyme states. In addition, although particular DNA sequences are known to interact specifically with RNAP, optical tweezers lacks the exact sequence registration, limiting methods of studying sequence - enzyme interactions at high resolution. At the University of Washington, our group has helped develop a new single-molecule technology uniquely suited to study the details of RNAP dynamics. This method, Single molecule Picometer Resolution Nanopore Tweezers (or SPRNT), is based off of the concept of nanopore DNA sequencing [5] and allows monitoring the translocation of single motor enzymes along DNA at unprecedented spatiotemporal resolution (40 picometers at millisecond time scales). In addition, each enzyme position measurement with SPRNT corresponds to a particular DNA sequence, allowing direct observation of enzyme interactions with DNA sequence. SPRNT has already been used to study motor proteins that walk along DNA: a DNA polymerase [6] [7] and a DNA helicase [8] [9]. In this thesis, I present my role in developing SPRNT to investigate RNAP during transcription. In section 0.1, I outline the relevant molecular systems and the techniques used to investigate them. I describe the development of SPRNT, starting with the fundamentals of nanopore sequencing, and compare this new technique to other methods. In section 0.2, I detail the methods developed to investigate E. coli RNAP with SPRNT, providing a guideline for future investigations of this enzyme. In section 0.3, I describe the initial results that were used to motivate and improve upon these methods. In section 0.4, I recount the relevant results of this investigation. I present the first measurements of single steps of E. coli RNAP during transcription at biologically relevant [NTPs] under an assisting force. I summarize how these measurements were used to track enzyme transitions between different states, developing a model for transcription. Using this model, I extrapolate the results obtained under various assisting forces to calculate the relevant rate constants at zero-force, providing a view into how RNAP behaves in its native environment. Next, I describe more SPRNT experiments that investigate RNAP pausing at particular sequences, including the Ơ̐1rst single-molecule detection of a 0half-translocated0 state during pausing. I detail a model for RNAP pausing resulting from this data. Finally, in section 0.5, I discuss the conclusions drawn from this work and the role SPRNT can play in future studies of transcription.

Download or read book Nucleic Acids Abstracts written by and published by . This book was released on 1994 with total page 1122 pages. Available in PDF, EPUB and Kindle. Book excerpt: