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The Role Of Spt5 Nucleic Acid Contacts In Promoter Proximal Pausing Of Rna Polymerase Ii
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Book Synopsis The Role of Spt5-nucleic Acid Contacts in Promoter Proximal Pausing of RNA Polymerase II. by : Roberta Dollinger
Download or read book The Role of Spt5-nucleic Acid Contacts in Promoter Proximal Pausing of RNA Polymerase II. written by Roberta Dollinger and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Promoter proximal pausing of RNA Polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires DSIF and NELF. DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. I used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. I identified two domains of Spt5, the KOW4 and NGN domains, that directly facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short alpha helical motif that is in close proximity to the non-transcribed DNA template strand. Mutation of this alpha helical motif in Drosophila has a male-specific dominant negative effect. The alpha helical motif is also needed to support fly viability. I also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex and that DSIF-Pol II association is independent of the nascent transcript. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, I show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex.
Book Synopsis Factors Controlling Promoter-proximal Pausing by RNA Polymerase II by : Nicholas James Fuda
Download or read book Factors Controlling Promoter-proximal Pausing by RNA Polymerase II written by Nicholas James Fuda and published by . This book was released on 2012 with total page 202 pages. Available in PDF, EPUB and Kindle. Book excerpt: Most gene expression is regulated at the level of transcription, and the transition from initiation to productive elongation is a key point of regulation. This transition is accompanied by pausing of transcriptionally engaged polymerase in the promoter-proximal region of several heat shock genes. Although this mechanism of regulation was long thought to be limited to a few genes, recent evidence has indicated that pausing is wide-spread in higher eukaryotes. Therefore, it is increasingly important to understand the mechanisms controlling the paused polymerase. I have investigated how the site of pausing on Hsp70 is specified using highresolution mapping of polymerase on reporter genes with shifted pausing site sequences. The results indicate that the downstream sequence dictates pause position and the overall level of pausing. I have also used RNAi knock-down in Drosophila cell culture to study the roles of several factors in establishing, maintaining, and releasing the paused polymerase. These experiments have shown GAGA factor is required for pausing on many of its target genes, and the knock-down effects indicate it is involved in establishing the pause. In contrast, Spt5, a protein previously shown to enhance pausing in vitro, reduces pausing genome-wide by increasing levels of elongating polymerase. Two kinases, P-TEFb and CDK12, function in productive elongation. Previously our lab showed that P-TEFb inhibition prevented the transition into elongation, limiting the polymerase to the 5' end of the heat shock-induced Hsp70 gene. I mapped these polymerases in high resolution to show they occupied sites further downstream than the normal pause sites, suggesting P-TEFb activity may not solely release the paused polymerase. I also determined the localization of CDK12 on active genes. Its localization downstream of P-TEFb suggests that these kinases may have distinct functions. Finally, I have examined the role of Fcp1 in Hsp70 transcription. Our lab previously showed the CTD phosphatase Fcp1 was required for optimum expression of Hsp70 mRNA. Fcp1 knock-down reduced the heat shock levels of Pol II and increased phosphorylation of nonchromatin bound Pol II, indicating that Fcp1 recycling of RNA polymerase II to an initiationcompetent form is required for optimal Hsp70 heat shock transcription.
Book Synopsis Regulation of Transcription Through RNA Polymerase II Promoter-proximal Pausing by : Melodi Damla Tastemel
Download or read book Regulation of Transcription Through RNA Polymerase II Promoter-proximal Pausing written by Melodi Damla Tastemel and published by . This book was released on 2018 with total page 100 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Studies on the Role of Enhancer RNAs in the Release of RNA Polymerase II from Promoter-proximal Pausing by : Vladyslava Gorbovytska
Download or read book Studies on the Role of Enhancer RNAs in the Release of RNA Polymerase II from Promoter-proximal Pausing written by Vladyslava Gorbovytska and published by . This book was released on 2021 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis RNA Exosome by : Torben Heick Jensen
Download or read book RNA Exosome written by Torben Heick Jensen and published by Springer Science & Business Media. This book was released on 2011-06-29 with total page 161 pages. Available in PDF, EPUB and Kindle. Book excerpt: The diversity of RNAs inside living cells is amazing. We have known of the more “classic” RNA species: mRNA, tRNA, rRNA, snRNA and snoRNA for some time now, but in a steady stream new types of molecules are being described as it is becoming clear that most of the genomic information of cells ends up in RNA. To deal with the enormous load of resulting RNA processing and degradation reactions, cells need adequate and efficient molecular machines. The RNA exosome is arising as a major facilitator to this effect. Structural and functional data gathered over the last decade have illustrated the biochemical importance of this multimeric complex and its many co-factors, revealing its enormous regulatory power. By gathering some of the most prominent researchers in the exosome field, it is the aim of this volume to introduce this fascinating protein complex as well as to give a timely and rich account of its many functions. The exosome was discovered more than a decade ago by Phil Mitchell and David Tollervey by its ability to trim the 3’end of yeast, S. cerevisiae, 5. 8S rRNA. In a historic account they laid out the events surrounding this identification and the subsequent birth of the research field. In the chapter by Kurt Januszyk and Christopher Lima the structural organization of eukaryotic exosomes and their evolutionary counterparts in bacteria and archaea are discussed in large part through presentation of structures.
Book Synopsis Molecular Mechanisms of Factors that Control RNA Polymerase II Transcription Elongation Dynamics by : Manchuta Dangkulwanich
Download or read book Molecular Mechanisms of Factors that Control RNA Polymerase II Transcription Elongation Dynamics written by Manchuta Dangkulwanich and published by . This book was released on 2015 with total page 137 pages. Available in PDF, EPUB and Kindle. Book excerpt: The expression of a gene begins by transcribing a target region on the DNA to form a molecule of messenger RNA. As transcription is the first step of gene expression, it is there- fore highly regulated. The regulation of transcription is essential in fundamental biological processes, such as cell growth, development and differentiation. The process is carried out by an enzyme, RNA polymerase, which catalyzes the addition of a nucleotide complementary to the template and moves along the DNA one base pair at a time. To complete its tasks, the enzyme functions as a complex molecular machine, possessing various evolutionarily designed parts. In eukaryotes, RNA polymerase has to transcribe through DNA wrapped around histone proteins forming nucleosomes. These structures represent physical barriers to the transcribing enzyme. In chapter 2, we investigated how each nucleosomal component--the histone tails, the specific histone-DNA contacts, and the DNA sequence--contributes to the strength of the barrier. Removal of the tails favors progression of RNA polymerase II into the entry region of the nucleosome by locally increasing the wrapping-unwrapping rates of the DNA around histones. In contrast, point mutations that affect histone-DNA contacts at the dyad abolish the barrier to transcription in the central region by decreasing the local wrapping rate. Moreover, we showed that the nucleosome amplifies sequence-dependent transcriptional pausing, an effect mediated through the structure of the nascent RNA. Each of these nucleosomal elements controls transcription elongation by distinctly affecting the density and duration of polymerase pauses, thus providing multiple and alternative mechanisms for control of gene expression by additional factors. During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme was proposed. In chapter 3, we challenged individual yeast RNA polymerase II (Pol II) with a nucleosome as a "road block", and separately measured the forward and reverse translocation rates with our single-molecule transcription elongation assay. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mech-anism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. This kinetic model provides a framework to study the influence of various factors on transcription dynamics. To further dissect the operation of Pol II, we focused on the trigger loop, a mobile element near the active site of the enzyme. Biochemical and structural studies have demonstrated that the trigger loop makes direct contacts with substrates and promotes nucleotide incorporation. It is also an important regulatory element for transcription fidelity. In chapter 4, we characterized the dynamics of a trigger loop mutant RNA polymerase to elucidate the roles of this element in transcription regulation, and applied the above kinetic framework to quantify the effects of the mutation. In comparison to the wild-type enzyme, we found that the mutant is more sensitive to force, faster at substrate sequestration, and more efficient to return from a pause to active transcription. This work highlighted important roles of regulatory elements in controlling transcription dynamics and fidelity. Moreover, RNA polymerase interacts with various additional factors, which add layers of regulation on transcription. Transcription factors IIS (TFIIS) and IIF (TFIIF) are known to interact with elongating RNA polymerase directly and stimulate transcription. In chapter 5, we studied the effects of these factors on elongation dynamics using our single molecule assay. We found that both TFIIS and TFIIF enhance the overall transcription elongation by reducing the lifetime of transcriptional pauses and that TFIIF also decreases the probability of pause entry. Furthermore, we observed that both factors enhance the efficiency of nucleosomal transcription. Our findings helped elucidate the molecular mechanisms of gene expression modulation by transcription factors. In summary, we have dissected the mechanisms by which the nucleosomal elements regulate transcription, and derived a quantitative kinetic model of transcription elongation in a linear Brownian ratchet scheme with the slow translocation of the enzyme. The corresponding translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states. This observation confers the enzyme its high propensity to pause, thus allowing additional regulatory mechanisms during pausing. TFIIS and TFIIF, for example, regulate transcription dynamics by shortening the lifetime of Pol II pauses. On the other hand, the trigger loop of Pol II regulates both the active elongation and pausing. These examples illustrate molecular mechanisms of cis- and trans-acting factors regulate the dynamics of transcription elongation.
Book Synopsis Fnctional Evolution of Promoter-proximal Pausing Factors in the Regulation of RNA Polymerase II Transcription by : Gregory T. Booth
Download or read book Fnctional Evolution of Promoter-proximal Pausing Factors in the Regulation of RNA Polymerase II Transcription written by Gregory T. Booth and published by . This book was released on 2018 with total page 404 pages. Available in PDF, EPUB and Kindle. Book excerpt: Promoter-proximal pausing of RNA Polymerase II (Pol II) is now recognized as a ubiquitous mechanism for regulating gene expression in metazoans. By capturing engaged Pol II shortly after transcription initiation, genes are primed for activation of RNA synthesis, enabling cells to rapidly alter global transcription programs. However, despite conservation of many factors involved in establishing this regulatory platform, many eukaryotes do not control gene expression through this process. Here, the examination of the global transcriptional landscape in two distantly related yeast revealed unprecedented divergence in Pol II distributions across genes. Previously undescribed pause-like profiles were identified within promoter-proximal regions of the fission yeast, Schizosaccharomyces pombe, that are sensitive to loss of the conserved elongation factor, Spt4. Thus, fission yeast might employ a variant of the system of regulation found in higher eukaryotes In flies and mammals, Pol II arrested within the promoter proximal region of a gene can only be released through the activity of a positive-transcription elongation factor (P-TEFb), composed of kinase (Cdk9) and cyclin (CycT1/2) subunits. Investigating the functional impact of Cdk9 on transcription in fission yeast revealed that, unlike most metazoan systems, Pol II in S. pombe is capable of overcoming the early elongation barrier after kinase inhibition, although not without consequence. However, fission yeast lack the metazoan-specific negative elongation factor complex (NELF) involved in pausing, perhaps limiting their ability to control the release of Pol II through phosphorylation of the elongation complex. Ultimately, by depleting pausing factors from cell lines derived from Drosophila melanogaster, it was tested whether NELF is required for P-TEFb-regulated pause escape. While global transcription is largely unaffected by the loss of NELF, upon inhibition of Cdk9, a significant amount of Pol II is aberrantly released from the pause, suggesting reduced control of this regulation. These findings suggest that NELF may have evolutionarily refined an ancestral promoter-proximal architecture of the transcription elongation complex, giving rise to a novel mechanism for gene regulation.
Book Synopsis Dissection of the Precise Mechanisms of RNA Polymerase II Pausing and Elongation Using Nascent Transcript Analysis by : Hojoong Kwak
Download or read book Dissection of the Precise Mechanisms of RNA Polymerase II Pausing and Elongation Using Nascent Transcript Analysis written by Hojoong Kwak and published by . This book was released on 2013 with total page 183 pages. Available in PDF, EPUB and Kindle. Book excerpt: Limiting RNA polymerase II (Pol II) at various stages of the transcription cycle is critical for gene regulation, which often occurs during the elongation stage at promoter proximal pause sites and in gene bodies. To determine the distribution of Pol II along genes, I used nascent transcript analysis as a general method. First, I identified the precise positions of Pol II pausing near promoters using a genome-wide nuclear run-on, called Precision Run-On sequencing (PRO-seq) in Drosophila embryonic cells. Using this, I revealed how the position of pausing is associated with initiation and promoter DNA elements. To further dissect the precise dynamics of paused Pol II, I probed the stability of paused Pol II and its termination by analyzing steady-state turn-over of the nascent transcript associated with Drosophila Hsp70 promoter. This shows that paused Pol II on Hsp70 is stable for around 5 min and can either terminate or elongate into the gene body, which is consistent with optical measurements of paused Pol II. I also examined how Pol II elongates during the time course of rapid and robust inhibition of pause escape in mouse embryonic stem cells. The analysis of the elongation rates in nearly 1,000 genes showed tight interplay between promoter proximal pausing, early elongation rates, and co-transcriptional splicing at the beginning of the genes. Finally, I demonstrate that the nascent transcriptome analysis methods can be directly extended into mammalian tissues, and show possibility of linking the study of the fundamental mechanism of Pol II into biomedical applications.
Download or read book HIV-1 Latency written by Guido Silvestri and published by Springer. This book was released on 2018-10-11 with total page 253 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume summarizes recent advances in understanding the mechanisms of HIV-1 latency, in characterizing residual viral reservoirs, and in developing targeted interventions to reduce HIV-1 persistence during antiretroviral therapy. Specific chapters address the molecular mechanisms that govern and regulate HIV-1 transcription and latency; assays and technical approaches to quantify viral reservoirs in humans and animal models; the complex interchange between viral reservoirs and the host immune system; computational strategies to model viral reservoir dynamics; and the development of therapeutic approaches that target viral reservoir cells. With contributions from an interdisciplinary group of investigators that cover a broad spectrum of subjects, from molecular virology to proof-of-principle clinical trials, this book is a valuable resource for basic scientists, translational investigators, infectious-disease physicians, individuals living with HIV/AIDS and the general public.
Book Synopsis Transcription and Splicing by : B. D. Hames
Download or read book Transcription and Splicing written by B. D. Hames and published by Oxford University Press, USA. This book was released on 1988 with total page 238 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book gives a co-ordinated review of our present knowledge of eukaryotic RNA synthesis.
Book Synopsis Chromatin Immunoprecipitation Assays by : Philippe Collas
Download or read book Chromatin Immunoprecipitation Assays written by Philippe Collas and published by Methods in Molecular Biology. This book was released on 2009-08-25 with total page 290 pages. Available in PDF, EPUB and Kindle. Book excerpt: In this book, researchers deeply involved in the development and improvement of chromatin immunoprecipitation assays (ChIP) provide cutting-edge protocols devoted to the most recent progress in ChIP and related subjects.
Book Synopsis Characterization of Novel DNA Elements Necessary for Sigma-dependent Promoter Proximal Transcriptional Pausing by : Jeremy Gilbert Bird
Download or read book Characterization of Novel DNA Elements Necessary for Sigma-dependent Promoter Proximal Transcriptional Pausing written by Jeremy Gilbert Bird and published by . This book was released on 2013 with total page 302 pages. Available in PDF, EPUB and Kindle. Book excerpt: Transcription is not necessarily uniform in rate, but instead consists of multiple periods of continuous elongation by RNA polymerase (RNAP) interrupted by occasional pausing events. These pauses have several determined origins, including the interaction of protein co-factors, the influence of transcribed RNA secondary structure, and a direct effect of the sequence composition of the DNA being transcribed. Promoter proximal pauses, in which RNAP pauses tens of nucleotides downstream of the transcription start site, have been observed in systems ranging from bacteria to more complex eukaryotes such as D. melanogaster and humans, and play important regulatory roles in organisms of all levels. In E. coli, [sigma]70-dependent promoter proximal pauses are of particular interest because lambdoid phage late gene promoters require these pauses for the loading of the anti-terminator Q protein, which is necessary for transcription of the phage late genes during lytic growth. The primary interaction that induces [sigma]70-dependent pauses is between the non-template strand of the -10-like sequence and [sigma]70 region 2. However, recent evidence identifies other important sequences including several nucleotides around the pause site itself that are required for pausing (Perdue and Roberts 2010). Mutational studies of lambdoid phage 82 promoter pR' show that a 3-bp GC-rich sequence primarily promotes pausing through the template DNA strand, but also acts through a non-template DNA strand interaction with [sigma]70. I show that this template strand sequence determines where the pause occurs. It is likely that in [sigma]-dependent promoterproximal pausing, the [sigma]70/DNA interaction anchors the elongation complex upstream of the pause site, requiring it to "scrunch" (by analogy to scrunching during initial transcription (Kapanidis et al. 2006)) and to create an enlarged DNA bubble as the active center reaches the pause site. "Scrunched" complexes are energetically strained, but stable at the pause site; my data suggest that the G/C-rich template sequence is a critical element in stabilizing the paused, scrunched structure. Mutational studies of the A/T-rich region demonstrate that the terminal nucleotide of the pause site plays an important role in pause formation, and that the base composition of the A/T-rich sequence determines the likelihood that a transcription complex escapes the pause. My data also suggest that a [sigma]70-dependent paused transcription complex enters the paused state through a pretranslocated state of the enzyme. Through these studies, I have expanded the current understanding of the protein/nucleotide and nucleotide/nucleotide interactions that constitute the [sigma]70dependent promoter proximal paused complex.
Book Synopsis Protein Phosphorylation in Human Health by : Cai Huang
Download or read book Protein Phosphorylation in Human Health written by Cai Huang and published by BoD – Books on Demand. This book was released on 2012-09-06 with total page 482 pages. Available in PDF, EPUB and Kindle. Book excerpt: 15 chapters on protein phosphorylation and human health written by expert scientists. Covers most important research hot points, such as Akt, AMPK and mTOR. Bridges the basic protein phosphorylation pathways with human health and diseases. Detailed and comprehensive text with excellent figure illustration.
Book Synopsis The Role of Downstream DNA in Promoter Escape by RNA Polymerase II by : Xiaoxue Wang
Download or read book The Role of Downstream DNA in Promoter Escape by RNA Polymerase II written by Xiaoxue Wang and published by . This book was released on 2000 with total page 184 pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis Role of Promoter DNA Sequences and Environmental Stress in Gene Regulation by RNA Polymerase II Pausing by : Parul Tomar
Download or read book Role of Promoter DNA Sequences and Environmental Stress in Gene Regulation by RNA Polymerase II Pausing written by Parul Tomar and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Book Synopsis RNA Polymerases as Molecular Motors by : Robert Landick
Download or read book RNA Polymerases as Molecular Motors written by Robert Landick and published by Royal Society of Chemistry. This book was released on 2021-11-23 with total page 295 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.
Download or read book Heat Shock Factor written by Akira Nakai and published by Springer. This book was released on 2016-01-06 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents a large amount of information related to the heat shock response and heat shock factor (HSF), describes core observations about molecular mechanisms and pathophysiological roles, and provides fundamental concepts on the basis of information from diverse aspects. This adaptive response to high temperature or protein misfolding is a fundamental mechanism to maintain the capacity of protein homeostasis, or proteostasis, and is evolutionally conserved among all living organisms, including bacteria and humans, on the earth. Furthermore, physiological and pathological roles of HSF have been extensively studied in fruit fly, worm, and mouse models. It has been revealed that HSF plays roles in development of the brain, reproductive and sensory organs, and in ageing, inflammation, and circadian rhythm. Analysis of the mechanisms have uncovered that HSF exerts a wide range of effects on gene expression and epigenetic status on the whole genome. Moreover, loss or gain of HSF function is also closely related to protein-misfolding diseases including neurodegenerative diseases, psychiatric diseases, heart diseases, and cancers. Therefore, HSF is now thought to be a promising therapeutic target for treatment of these refractory diseases. For undergraduate students, this is a highly understandable source of information on heart shock response and HSF, covering the basis of HSF biology, the physiological role of HSF, and disease associated with HSF function. This book not only serves as a guide to the heat shock response and HSF for students and young researchers in other fields, but also is a cornerstone for future work in the field related to the heat shock response and HSF.
