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 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 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 on Transcriptional Elongation in Prokaryotes and Eukaryotes written by Jing Zhou and published by . This book was released on 2012 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Transcription, the process of copying genetic information stored in DNA into RNA, is fundamental to life. It is carried out by an extraordinary nano-machine called RNA polymerase (RNAP). Transcriptional elongation, during which RNAP moves along the DNA, adding one nucleotide at a time to the RNA transcript, is highly dynamic and regulated. The motion of RNAP is discontinuous and interrupted by pauses that play an essential role in gene regulation. Fundamental questions regarding the mechanisms of elongation and its modulation by transcription factors, however, remain unclear. In this dissertation, I focus on using high-resolution, optical trapping techniques to study the mechanisms of transcriptional elongation by both prokaryotic and eukaryotic RNA polymerases at the single-molecule level. First, I describe the studies on how the motion of single E.coli RNAP molecules is modulated by two universally conserved, essential transcription factors (NusA and NusG). From individual transcriptional elongation records, the rates of entering pause states, the pause state lifetimes, and the pause-free elongation speeds can all be extracted. By studying the effects of NusA (and NusG) on these kinetic rates as a function of the applied load, we were able to develop a quantitative kinetic scheme for elongation and pausing. This model not only explains the functions of NusA/NusG, but also provides insight into the mechanism of transcriptional pausing, which had previously been controversial. Second, a novel optical-trapping assay capable of directly probing elongation by individual eukaryotic RNA polymerase II (RNAPII) molecules will be described. We find that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, not only affects each of the three main phases of elongation, namely: substrate binding, translocation, and catalysis; but also plays a critical role in controlling the fidelity of transcription. Our data also support a Brownian ratchet model for elongation which incorporates a secondary nucleotide binding site.

Download or read book Single Molecule Studies of Nucleic Acids and Their Proteins written by David Bensimon and published by Oxford University Press. This book was released on 2018-11-30 with total page 224 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book provides the basis for understanding the elastic properties of nucleic acids (DNA, RNA), the methods used to manipulate them (e.g. optical, magnetic and acoustic tweezers and traps), and how to observe their interactions with proteins (e.g. fluorescence microscopy, FCS, FRET, etc.). It then exemplifies the use of these various methods in the study of three families of DNA enzymes: polymerases, helicases and topoisomerases. The book aims not to be exhaustive, but rather to stimulate the imagination of readers in the application of these single molecule approaches to the study of DNA/RNA and their interactions.

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 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 RNA Polymerases as Molecular Motors written by Henri Buc and published by Royal Society of Chemistry. This book was released on 2009-03-10 with total page 353 pages. Available in PDF, EPUB and Kindle. Book excerpt: The cell can be viewed as a 'collection of protein machines' and understanding these molecular machines requires sophisticated cooperation between cell biologists, geneticists, enzymologists, crystallographers, chemists and physicists. To observe these machines in action, researchers have developed entirely new methodologies for the detection and the nanomanipulation of single molecules. 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 speciality. 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. For more than thirty years, the study of RNA polymerases has benefited from intense cooperation between the scientific partners involved in the various fields listed above. It is hoped that a collection of essays from outstanding scientists on this subject will catalyse the convergence of scientific efforts in this field, as well as contribute to better teaching at advanced levels in Universities.

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 Single Molecule and Synthetic Biology Studies of Transcription written by Bradley Michael Zamft and published by . This book was released on 2011 with total page 326 pages. Available in PDF, EPUB and Kindle. Book excerpt: The horizons of biology are ever expanding, from the discernment of the detailed mechanisms of enzyme function, to the manipulation of the physiological processes of whole organisms and ecosystems. Single molecule studies allow for the characterization of the individual processes that comprise an enzyme's mechanochemical cycle. Through standardization and generalization of biological techniques, components, and knowledge, synthetic biology seeks to expand the scale of biological experiments and to usher in an age of biology as a true engineering science, in which those studying different hierarchical levels of sophistication need not start from the fundamental biochemical principles underlying all biological experiments. Here we report our findings on the processes governing transcription and its role in gene expression through the use of both single molecule and synthetic biology methods. We have established a promoter-free, factor-free method of initiation of transcription by the mitochondrial RNA polymerase in Saccharomyces cerevisiae, Rpo41 through the use of synthetic oligonucleotides to imitate the hybridization geometry of Rpo41 during active transcription. Using this system, we have established that a sub-micromolar NTP concentration is appropriate for non-saturating transcriptional runoff assays. We have optimized the transcription buffer and found that 10 mM MgCl2, 40 mM KCl, and 10 mM DTT are sufficient for robust transcription. Stability studies show that Rpo41 loses approximately 30% of its activity during each freeze-thaw cycle, and that the pre-formed elongation complex loses transcriptional activity with a half-life of 7.4±1.5 hr. Through the use of optical trapping techniques, we have established a method to monitor the transcription of individual Rpo41 molecules in real time. This has allowed us to measure the kinetic rates of nucleotide incorporation by the enzyme: Km = 22±13 μM-1 and vmax = 25±2.5 bp/s. Both of these rates are more similar to those of the main nuclear RNA polymerase in the same organism, RNA Polymerase II (Pol II) than to that of the T7 RNA polymerase, despite the fact that Rpo41 is a single-subunit RNA polymerase with homology to those of the T-odd bacteriophage and no discernable homology to Pol II. Furthermore, like Pol II and the E. coli RNA polymerase, transcription by Rpo41 consists of periods of processive transcription interspersed with periods of pausing. We have also observed retrograde motion of Rpo41 during pauses, termed backtracking, a process that has not been reported in phage-like RNA polymerases. We have performed single molecule assays of transcription by both Pol II and Rpo41 on templates of differing base pair composition and found that, in general, the characteristics of pausing are attenuated in templates of higher GC content. Specifically, the frequency of pausing is decreased in GC-rich templates, as is the average pause duration. The distribution of pause durations is correspondingly shifted to shorter pauses on GC-rich templates. We discuss two mechanisms by which template composition may affect pausing: (1) movement of the backtracked transcription bubble is affected by differences in the base stacking energies from the disrupted/created DNA/DNA and RNA/DNA base pairs at the ends of the bubble, and (2) secondary structure of the nascent RNA upstream of the backtracked transcription bubble imposes an energetic barrier to its backward movement. We give in silico evidence that it is the latter mechanism. Incorporation of this secondary structure energy barrier (an "energy penalty") into a model of transcriptional pausing by backtracking allows for statistical fits of the mean pause densities, mean pause durations, and the distribution of pause durations for each enzyme on each template. Furthermore, incorporation of the energy penalty allows for fitting of the pause characteristics for a given enzyme using a single, enzyme specific hopping rate, k0, that is independent of template, and a single, template dependent energy penalty term, [Delta]GRNA, which is enzyme independent. For Rpo41, we find that k0, the hopping rate of the backtracked enzyme along DNA without RNA secondary structure, is 5.4±1.8 s-1, while it is 2.9±0.3 s-1 for Pol II. Furthermore, the average energy penalty due to the nascent RNA, [Delta]GRNA, on the AT-rich template used in this study is 0.7±0.1 kT, while it is 0.8±0.1 kT for random DNA and 1.0±0.1 kT for GC-rich DNA. In order to confirm that it is the secondary structure of the RNA that is the cause of the energy penalty, we performed the same single-molecule transcription assays in the presence of RNase A, an enzyme that digests unprotected RNA in both single-stranded and double-stranded form. The pausing characteristics of all traces on all templates in the presence of RNase A are statistically indistinguishable from those on AT-rich DNA without RNase, indicating that the RNase digested enough of the nascent RNA to disrupt any secondary structure. Protection of the 5' region of the nascent RNA by steric interactions between the polymerase and the RNase prevented full degradation of the RNA, and thus allowed for some backtracking. This strongly supports the new model, presented here, of modulation of transcriptional pausing by secondary structure of the nascent RNA. In contrast to the detailed and isolated nature of single-molecule transcription, we also performed a synthetic biology project involving Rpo41. The intent of this project was to investigate the plausibility of the creation of a transcriptionally independent mitochondrion, and by extension a minimal cell, by movement of the mitochondrial transcriptional machinery from the nuclear to the mitochondrial genome. Thus we performed in vivo mitochondrial transformation of yeast cells with a synthetic construct containing the gene encoding for Rpo41. We report that we have successfully integrated said synthetic gene into the mitochondrial genome, and have seen its expression to the transcriptional level. Furthermore, we are fairly confident that the full, intact mRNA of the synthetic gene is being created within the mitochondrial matrix. We have not been able to detect expression of the protein product of the integrated synthetic construct, nor have we been able to isolate a strain that exhibits its expression in the absence of the wild-type, nuclear copy. Because the length of Rpo41 is longer than any other protein synthesized within the mitochondrial organelle, we have begun experiments to determine the maximal polypeptide length able to be translated by the mitochondrial ribosome and associated cofactors.

Download or read book Single Molecule Studies of Bacterial Transcription Under Force and Torque Using Optical Tweezers written by Yara Xochitl Mejia and published by . This book was released on 2009 with total page 282 pages. Available in PDF, EPUB and Kindle. Book excerpt: The present dissertation uses Optical Tweezers to examine the inner workings of one of the most relevant molecular motors in the cell: RNA polymerase. E. coli RNA polymerase has been studied for almost half a century, but fundamental questions about its translocation mechanism along DNA, as well as its pausing and stalling behavior still remain. Due to RNAP's heterogeneous behavior, a single molecule approach presents unique advantages. As part of this work, single molecule transcription experiments were done at temperatures between 7°C and 45°C. Within this temperature range, the pause-free velocity of RNAP increases with temperature with an activation energy of 9.7 ± 0.7 kcal/mole. Moreover, temperature affects pause entry and the stalling force, but not pause duration. This dissertation also presents the first single molecule study of Trigger Loop (TL) mutants, a domain thought to have a crucial role in enzyme catalysis. Our results identify TL folding as a rate-determining step in elongation and correlate TL helix propensity with pause-free velocity. Based on the inverse relation between pause-free velocity and pause entry for the mutant and wildtype polymerases and for transcription with nucleotide analogs, a quantitative kinetic model was constructed. An analysis of pause durations indicated that the TL has no role in pause recovery. Furthermore, a novel single molecule assay was developed to study RNAP's rotation velocity during elongation and in response to torsional load. Here, the DNA is stretched between two beads, and a "rotor bead" of different sizes is attached to the rotating polymerase. The pause-free angular speed is seen to decrease for increasing rotational loads corresponding to a constant torque value of 7 pNnm. Further analysis demonstrates that RNAP acts as a Brownian Ratchet and exerts an average energy per step of 1 KBT. Rotational load does not, however, have an effect on pause entry or duration. Finally, I describe a novel technique for modifying the twist of DNA. This Hybrid Tweezers technique was used for the formation of DNA plectonemes and braids, as well as for studying transcription under Torque. Together, these experiments have constructed a clearer picture of the kinetics, energetics and mechanisms of RNAP.

Download or read book A Single Molecule Study of RNA Polymerase and Tata box Binding Protein written by S.F. Tolic-Nørrelykke and published by . This book was released on 2002 with total page 120 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book RNA Polymerase III Transcription written by Robert J. White and published by Springer Science & Business Media. This book was released on 2013-11-11 with total page 274 pages. Available in PDF, EPUB and Kindle. Book excerpt: This monograph reviews and summarizes the substantial body of work that has been published on the transcription by polymerase III over the past 5 years. Progress in this field has been very rapid since 1993, and this new edition incorporates all the recent developments and offers the reader a highly detailed analysis of the current state of research on this largest and most complex of the eukaryotic RNA polymerases.

Download or read book RNA as Molecular Motors 2E written by Robert Landick and published by Royal Society of Chemistry. This book was released on 2021-12-15 with total page 295 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book, written by expert scientists in the field, analyses how these diverse fields of research interact on a specific example - RNA polymerase.

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 Investigation of Bacterial Transcription Using Single Molecule Techniques written by SangYoon Chung and published by . This book was released on 2016 with total page 124 pages. Available in PDF, EPUB and Kindle. Book excerpt: The numerous complex molecular processes occurring inside living cells are primarily carried out by proteins and other biopolymers, such as ribonucleic acids (RNA). The identity and quantity of the different proteins and RNA determine the cell's phenotype and changes in response to the environment. Therefore, the internal composition of the cell in terms of the type and concentration of proteins and RNA is tightly regulated. Gene expression is the process of using the DNA sequence information to produce these biopolymers. Transcription, the initial step in gene expression, where one strand of DNA is used as template by the enzyme RNA polymerase (RNAP) for synthesizing a complementary RNA or transcript. Since cell phenotype is mostly determined by transcription, a complex regulatory mechanism exists involving a large number of factors to control the level of transcription of a gene. Although most studies are focused on multiple cycles of either transcription or association of DNA and RNA Polymerase (RNAP) to make RNAP-Promoter open complex (RPO), single round transcription studies are crucial in elucidating the mechanism of sophisticated RNAP-DNA interactions and its kinetics in transcription. In this context, we have developed a novel in vitro quenching based single round transcription assay using single molecule detection. Using this, we could successfully dissect initiation kinetics starting from different initial transcribing stages and found that transcription initiation doesn't follow a sequential model (as commonly believed). Instead, we identified a previously uncharacterized state that is unique to initial transcribing complexes and associated with the backtracked RNAP-DNA complex. Also, we have investigated the size/concentration effects of various osmolytes and macromolecular crowding agents, which mimic the crowded cellular environment, on actively-transcribing RNAP and found enhancement in transcription kinetics by larger crowding agents at the same viscosity.

Download or read book Single Molecule Studies of Bacterial RNA Polymerase Dynamics written by Timothy T. Harden and published by . This book was released on 2016 with total page 107 pages. Available in PDF, EPUB and Kindle. Book excerpt: