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Author:
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 145
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Transcription involves many reaction steps and intermediates. Many phenomena in transcription kinetics are covered by ensemble average. Single-molecule DNA nanomanipulation techniques uncover these transcription kinetic events via determination of a transcription bubble in real time. In this dissertation, we focus on the transcription kinetics of bacterial RNAP. The study of transcription kinetics in this thesis can be divided into 4 main subjects: 1) The study of abortive initiation mechanism: Through a single-molecule DNA nanomanipulation technique, we tested the three models proposed for the mechanism of abortive initiation - inchworming, scrunching and transient excursion - on T5 N25 promoter. Of the three models, only the scrunching model involves a change in the size of the transcription bubble during abortive initiation, which was observed by single-molecule DNA nanomanipulation technique. 2) The study of the kinetics of elongation and termination: By introducing varying transcribed region lengths into DNA templates, the kinetics of elongation and terminator rewinding were studied. The resulting kinetics, determined by the single-molecule nanomanipulation technique, was analyzed by different regression methods. In the normal regression, the elongation velocity is 10 b/s and terminator rewinding takes 3.4 s on a tR2 terminator. Contrarily, through Poisson regression, the elongation velocity ranges from 6.4 b/s to 12.5 b/s and terminator rewinding takes 11.9 s on a tR2 terminator. 3) The study of a promoter sequence's effect: The effect from sequence of the T5 N25 promoter and T5 N25 antiDSR promoter in transcription was evaluated. The transcription bubble sizes of open complex and initial transcribing complex using the T5 N25 antiDSR promoters are larger than the ones from the T5 N25 promoter. The difference in the two promoter sequences does not have an effect on the transcription bubble size of an elongation complex. And elongation and terminator rewinding kinetics are not affected. On the other hand, abortive initiation and promoter escape are affected by the difference in the promoter sequence. 4) The study of the effect of transcription factor-GreB: The effect of transcription factor-GreB on abortive initiation was evaluated by the single-molecule DNA nanomanipulation technique. GreB does not affect the transcription bubble size during abortive initiation, but does reduce the lifetime of initial transcribing complex.
Author:
Publisher:
ISBN:
Category : DNA polymerases
Languages : en
Pages : 145
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Book Description
Transcription involves many reaction steps and intermediates. Many phenomena in transcription kinetics are covered by ensemble average. Single-molecule DNA nanomanipulation techniques uncover these transcription kinetic events via determination of a transcription bubble in real time. In this dissertation, we focus on the transcription kinetics of bacterial RNAP. The study of transcription kinetics in this thesis can be divided into 4 main subjects: 1) The study of abortive initiation mechanism: Through a single-molecule DNA nanomanipulation technique, we tested the three models proposed for the mechanism of abortive initiation - inchworming, scrunching and transient excursion - on T5 N25 promoter. Of the three models, only the scrunching model involves a change in the size of the transcription bubble during abortive initiation, which was observed by single-molecule DNA nanomanipulation technique. 2) The study of the kinetics of elongation and termination: By introducing varying transcribed region lengths into DNA templates, the kinetics of elongation and terminator rewinding were studied. The resulting kinetics, determined by the single-molecule nanomanipulation technique, was analyzed by different regression methods. In the normal regression, the elongation velocity is 10 b/s and terminator rewinding takes 3.4 s on a tR2 terminator. Contrarily, through Poisson regression, the elongation velocity ranges from 6.4 b/s to 12.5 b/s and terminator rewinding takes 11.9 s on a tR2 terminator. 3) The study of a promoter sequence's effect: The effect from sequence of the T5 N25 promoter and T5 N25 antiDSR promoter in transcription was evaluated. The transcription bubble sizes of open complex and initial transcribing complex using the T5 N25 antiDSR promoters are larger than the ones from the T5 N25 promoter. The difference in the two promoter sequences does not have an effect on the transcription bubble size of an elongation complex. And elongation and terminator rewinding kinetics are not affected. On the other hand, abortive initiation and promoter escape are affected by the difference in the promoter sequence. 4) The study of the effect of transcription factor-GreB: The effect of transcription factor-GreB on abortive initiation was evaluated by the single-molecule DNA nanomanipulation technique. GreB does not affect the transcription bubble size during abortive initiation, but does reduce the lifetime of initial transcribing complex.
Author: Andrey Revyakin
Publisher:
ISBN:
Category :
Languages : en
Pages : 510
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Author: SangYoon Chung
Publisher:
ISBN:
Category :
Languages : en
Pages : 124
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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: Jun Fan
Publisher:
ISBN:
Category :
Languages : en
Pages : 0
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The DNA in living cells is constantly threatened by damages from both endogenous and exogenous agents, which can threaten genomic integrity, block processes of replication, transcription and translation and have also genotoxic effects. In response to the DNA damage challenge, organisms have evolved diverse surveillance mechanisms to coordinate DNA repair and cell-cycle progression. Multiple DNA repair mechanisms, discovered in both prokaryotic and eukaryotic organisms, bear the responsibility of maintaining genomic integrity; these mechanisms include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and double strand break repair (DSBR). Transcription-coupled DNA repair (TCR) is a specialized NER subpathway characterized by enhanced repair of the template strand of actively transcribed genes as compared to the classical global genome repair (GGR) subpathway of NER which does not distinguish between template and non-template strands. TCR achieves specialization via the involvement of RNA polymerase (RNAP) and the Mfd (Mutation Frequency Decline) protein, also known as TRCF (transcription repair coupling factor). TCR repair initiates when RNAP stalls at a DNA lesion on the transcribed strand and serves as the da mage sensor. The stalled RNAP must be displaced so as to make the lesion accessible to downstream repair components. E. coli Mfd translocase participates in this process by displacing stalled RNAP from the lesion and then coordinating assembly of the UvrAB(C) components at th( damage site. Recent studies have shown that after binding to and displacing stalled RNAP, Mfd remains on the DNA in the form of a stable, translocating complex with evicted RNAP. So as to understand how UvrAB(C) are recruited via the Mfd-RNAP complex, magnetic trapping of individual, damaged DNA molecules was employed to observe-in real-time this multi¬component, multi-step reaction, up to and including the DNA incision reaction by UvrC. It was found that the recruitment of UvrA and UvrAB to the Mfd-RNAP complex halts the translocating complex and then causes dissolution of the complex in a molecular "hand-off" with slow kinetics Correlative single-molecule nanomanipulation and fluorescence further show that dissolution of the complex leads to loss of not only RNAP but also Mfd. Hand-off then allows for enhanced incision of damaged DNA by the UvrC component as compared to the equivalent single-moleculE GGR incision reaction. A global model integrating TCR and GGR components in repair was proposed, with the overall timescales for the parallel reactions provided.
Author: Bradley Michael Zamft
Publisher:
ISBN:
Category :
Languages : en
Pages : 326
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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:
Publisher:
ISBN:
Category : Dissertations, Academic
Languages : en
Pages : 800
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Author: Henri Buc
Publisher: Royal Society of Chemistry
ISBN: 1847559980
Category : Science
Languages : en
Pages : 353
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Book Description
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.
Author: Hua Yu
Publisher:
ISBN:
Category :
Languages : en
Pages : 306
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Author: Jing Tang (Ph. D.)
Publisher:
ISBN:
Category :
Languages : en
Pages : 147
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Rapid genome characterization is one of the grand challenges of genome science today. Although the complete sequences of certain representative human genomes have been determined, genomes from a much larger number of individuals are yet to be studied in order to fully understand genome diversity and genetic diseases. While current state-of-the-art sequencing technologies are limited by the large timescale and cost required to analyze a single sample, an alternative strategy termed DNA mapping has recently received considerable attention. Unlike sequencing which produces single-base resolution, DNA mapping resolves coarse-scale (~kbp) information of the sequence, which is much faster and cheaper to obtain, but still sufficient to discern genomic differences among individuals within a given species. Advances in fluorescence microscopy have allowed the possibility to directly map a single DNA molecule. This concept, though straightforward, faces a major challenge that the entropic tendency of polymeric DNA to adopt a coiled conformation must be overcome so as to optically determine the position of specific sequences of interest on the DNA backbone. The ability to control and manipulate the conformation of single DNA molecules, especially, to stretch them into a linear format in a consistent and uniform manner, is thus crucial to the performance of such mapping devices. The focus of this thesis is to develop a reliable single DNA stretching device that can be used in single molecule DNA mapping, and to experimentally probe the fundamental physics that govern DNA deformation. In the aspect of device design, the strategy we pursue is the use of an elongational electric field with a stagnation point generated in the center of a cross-slot or T channel to stretch DNA molecules. The good compatibility of electric field with small channel dimensions allows us to use micro- or nano-fabricated channels with height on the order of or smaller than the natural size of DNA to keep the molecule always in focus, a feature desirable for any mapping applications. The presence of the stagnation point allows the possibility to dynamically trap and stretch single DNA molecules. This trapping capability ensures uniform stretching within a sample ensemble, and also allows prolonged imaging time to obtain accurate detection results. We primarily investigate the effects of channel height on the stretching process, specifically, we seek the possibility of utilizing slit-like nanoconfinement to aid DNA stretching. Although extensive previous studies have demonstrated that geometric confinement of DNA can substantially alter the conformation and dynamics of these molecules at equilibrium, no direct studies of this non-equilibrium stretching process in confinement exist prior to the work presented in this thesis. We find that slit-like confinement indeed facilitates DNA stretching by reducing the deformation Abstract rate required to achieve a certain extension. However, due to the fact that the steric interactions between the DNA and the confining walls vanish at large extensions, highly stretched DNA under confinement behaves qualitatively similar to unconfined DNA except with screened hydrodynamic interactions, and a new time scale arises that should be used to describe the large change in extension with applied deformation rate. In a consecutive study, we examine the low-extension stretching process and observe a strongly modified coil-stretch transition characterized by two distinct critical deformation rates for DNA in confinement, different from the unconfined case where a single critical deformation rate exists. With kinetic theory modeling, we demonstrate that the two-stage coilstretch transition in confinement is induced by a modified spring force law, which is essentially related to the extension-dependent steric interactions between DNA and the confining walls. We also study aspects of the equilibrium conformation and dynamics of DNA in slit-like confinement in order to provide insight into regimes where existing studies show inconsistent results. We use both experiments and simulations to demonstrate that the in-plane radius of gyration and the 3D radius of gyration of DNA behaves differently in weak confinement. In strong confinement, we do not identify any evident change in the scalings of equilibrium size, diffusivity, and longest relaxation time of the DNA with channel height from the de Gennes regime to the Odijk regime. Although the transition between the de Gennes and Odijk regimes in slit-like confinement still remains an open question, our finding adds more experimental evidence to the side of a continuous transition. The impact of this thesis will be two-fold. We design a DNA stretching device that is readily applicable to single molecule DNA mapping and establish guidelines for the effective operation of the device. Our fundamental results regarding both the equilibrium and non-equilibrium dynamics of DNA molecules in slit-like confinement will serve as a solid basis for both the design of future devices aiming to exploit confinement to manipulate biopolymers, and more complicated studies of confined polymer physics.
Author: Mónica Fernández Sierra
Publisher:
ISBN:
Category :
Languages : en
Pages : 348
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