Single-molecule Studies of Transcription Initiation PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Single-molecule Studies of Transcription Initiation PDF full book. Access full book title Single-molecule Studies of Transcription Initiation by Diego Armando Duchi Llumigusin. Download full books in PDF and EPUB format.
Author: Diego Armando Duchi Llumigusin
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
Author: Diego Armando Duchi Llumigusin
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
Author: Kevin Oliver Kramm
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
Author: SangYoon Chung
Publisher:
ISBN:
Category :
Languages : en
Pages : 124
Get Book Here
Book Description
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.
Author: David Bensimon
Publisher: Oxford University Press
ISBN: 0192559966
Category : Science
Languages : en
Pages : 224
Get Book Here
Book Description
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.
Author: Bradley Michael Zamft
Publisher:
ISBN:
Category :
Languages : en
Pages : 326
Get Book Here
Book Description
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.
Author: Robert Landick
Publisher: Royal Society of Chemistry
ISBN: 1839160667
Category : Science
Languages : en
Pages : 295
Get Book Here
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
Get Book Here
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: Achim P. Popp
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
Author: Jing Zhou
Publisher:
ISBN:
Category :
Languages : en
Pages :
Get Book Here
Book Description
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.
Author:
Publisher:
ISBN: 9780815332183
Category : Cells
Languages : en
Pages : 0
Get Book Here
Book Description
