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The Scandoval. As you know we are in the middle of it, but did you ever stop to ask when was the first sign of something inappropriate going on between Tom and Raquel. Today, return guests, famed dress designer, Pol' Atteu, and husband Patrik Simpson. break down everything they saw as guests at good friend Scheana Shay's wedding. Patrik and Pol detail how they saw Tom Sandoval kissing Raquel. Yes, Sandoval. They break down how Producers must have known as they were present at Scheana's wedding filming 24/7, what they saw between the two, how they saw the “set up” Schwartz and Raquel kiss happening and much, much more. The more the boys talked, the more questions we had about how many times they saw this, what they saw, producers, the VPR cast and much, much more. Loved this chat? Feel free to check out Patrik and Pol on their Number One Fashion Pod, “Undressed”! Part Two Begins Now! @polatteu @patriksimpson @behindvelvetrope @davidyontef BONUS & AD FREE EPISODES Available at - www.patreon.com/behindthevelvetrope BROUGHT TO YOU BY: NEBULA9 VODKA - nebula9vodka.com (Use Promo Code VELVET For 10% Off Your Order) MICRODOSE- microdose.com (Use Code VelvetRope For Free Shipping and 30% Off Your First Order) ADVERTISING INQUIRIES - Please contact David@advertising-execs.com MERCH Available at - https://www.teepublic.com/stores/behind-the-velvet-rope?ref_id=13198 Learn more about your ad choices. Visit megaphone.fm/adchoices
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535882v1?rss=1 Authors: Ciotta, G., Singh, S., Gupta, A., Torres, D. C., Fu, J., Choudhury, R., Chu, W. K., Choudhary, C., Gahurova, L., Al-Fatlawi, A., Schroeder, M., Aasland, R., Poetsch, A., Anastassiadis, K., Stewart, A. F. Abstract: SETD1A is the histone 3 lysine 4 (H3K4) methyltransferase central to the mammalian version of the highly conserved eight subunit Set1 complex (Set1C) that apparently conveys H3K4 trimethylation (H3K4me3) onto all active Pol II promoters. Accordingly, mouse embryonic stem cells (ESCs) die when SETD1A is removed. We report that death is accompanied by loss of expression of DNA repair genes and accumulating DNA damage. BOD1L and BOD1 are homologs of the yeast Set1C subunit, Shg1, and subunits of the mammalian SETD1A and B complexes. We show that the Shg1 homology region binds to a highly conserved central a-helix in SETD1A and B. Like mutagenesis of Shg1 in yeast, conditional mutagenesis of Bod1l in ESCs promoted increased H3K4 di- and tri-methylation but also, like loss of SETD1A, loss of expression of DNA repair genes, increased DNA damage and cell death. In contrast to similar losses of gene expression, the converse changes in H3K4 methylation implies that H3K4 methylation is not essential for expression of the DNA repair network genes. Because BOD1L becomes highly phosphorylated after DNA damage and acts to protect damaged replication forks, the SETD1A complex and BOD1L in particular are key nodes for the DNA damage repair network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
In this episode of the Epigenetics Podcast, we caught up with Marcela Sjöberg from the University of Chile to talk about her work on the hydroxymethylation landscape in immune cells. At the beginning of her career Marcela Sjöberg worked on Polycomb and how modifications placed by this complex modulate the binding of RNA Pol II. Later, her focus shifted to hydroxymethylated cytosine and how it is involved in the inheritance of Metastable Epialleles in mouse. More recently, the laboratory is interested in transcription factor binding motifs and how hydroxymethylation of those binding motifs modulates the binding and activity of the respective transcription factors. References Sabbattini, P., Sjoberg, M., Nikic, S., Frangini, A., Holmqvist, P.-H., Kunowska, N., Carroll, T., Brookes, E., Arthur, S. J., Pombo, A., & Dillon, N. (2014). An H3K9/S10 methyl-phospho switch modulates Polycomb and Pol II binding at repressed genes during differentiation. Molecular Biology of the Cell, 25(6), 904–915. https://doi.org/10.1091/mbc.e13-10-0628 Kazachenka, A., Bertozzi, T. M., Sjoberg-Herrera, M. K., Walker, N., Gardner, J., Gunning, R., Pahita, E., Adams, S., Adams, D., & Ferguson-Smith, A. C. (2018). Identification, Characterization, and Heritability of Murine Metastable Epialleles: Implications for Non-genetic Inheritance. Cell, 175(5), 1259-1271.e13. https://doi.org/10.1016/j.cell.2018.09.043 Westoby, J., Herrera, M.S., Ferguson-Smith, A.C. et al. Simulation-based benchmarking of isoform quantification in single-cell RNA-seq. Genome Biol 19, 191 (2018). https://doi.org/10.1186/s13059-018-1571-5 Viner, C., Johnson, J., Walker, N., Shi, H., Sjöberg, M., Adams, D. J., Ferguson-Smith, A. C., Bailey, T. L., & Hoffman, M. M. (2016). Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet [Preprint]. Bioinformatics. https://doi.org/10.1101/043794 Related Episodes The Role of DNA Methylation in Epilepsy (Katja Kobow) DNA Methylation and Mammalian Development (Déborah Bourc'his) Effects of DNA Methylation on Chromatin Structure and Transcription (Dirk Schübeler) Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on LinkedIn Active Motif on Facebook Email: podcast@activemotif.com
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.375329v1?rss=1 Authors: Tomko, E. J., Luyties, O., Rimel, J. K., Tsai, C.-L., Fuss, J. O., Fishburn, J., Hahn, S., Tsutakawa, S. E., Taatjes, D. J., Galburt, E. A. Abstract: The general transcription factor TFIIH contains three ATP-dependent catalytic activities. TFIIH functions in nucleotide excision repair primarily as a DNA helicase and in Pol II transcription initiation as a dsDNA translocase and protein kinase. During initiation, the XPB/Ssl2 subunit of TFIIH couples ATP hydrolysis to dsDNA translocation facilitating promoter opening and the kinase module phosphorylates the C-terminal domain to facilitate the transition to elongation. These functions are conserved between metazoans and yeast; however, yeast TFIIH also drives transcription start-site scanning in which Pol II scans downstream DNA to locate productive start-sites. The ten-subunit holo-TFIIH from S. cerevisiae has a processive dsDNA translocase activity required for scanning and a structural role in scanning has been ascribed to the three-subunit TFIIH kinase module. Here, we assess the dsDNA translocase activity of ten-subunit holo- and core-TFIIH complexes (i.e. seven subunits, lacking the kinase module) from both S. cerevisiae and H. sapiens. We find that neither holo nor core human TFIIH exhibit processive translocation, consistent with the lack of start-site scanning in humans. Furthermore, in contrast to holo-TFIIH, the S. cerevisiae core-TFIIH also lacks processive translocation and its dsDNA-stimulated ATPase activity was reduced ~5-fold to a level comparable to the human complexes, potentially explaining the reported upstream shift in start-site observed in the absence of the S. cerevisiae kinase module. These results suggest that neither human nor S. cerevisiae core-TFIIH can translocate efficiently, and that the S. cerevisiae kinase module functions as a processivity factor to allow for robust transcription start-site scanning. Copy rights belong to original authors. Visit the link for more info
This week’s episode includes author Allan Sniderman and Associate Editor Anand Rohatgi as they discuss the expected 30-year benefits of early versus delayed primary prevention of cardiovascular disease by lipid lowering and management. TRANSCRIPT: Dr Greg Hundley: Welcome everyone to this September 1 issue, as we start into the fall in North America, and I guess we're getting into spring-ish in the Southern hemisphere. Today, it's just myself, Dr Greg Hundley, Associate Editor and Director of the Pauley Heart Center at VCU Health in Richmond, Virginia. And, I'm so sad because my good friend, Carolyn, cannot be with us today. However, we have a great feature for the latter half of this recording and discussing some of the benefits of primary prevention using lipid lowering therapy to help prevent cardiovascular disease. But, before we get to that, let's grab a cup of coffee and let's go through some of the other articles in this issue. So, the first one is from the world of basic science and it's from Professor Eldad Tzahor, from the Weizmann Institute of Science. And, it's focusing on Agrin. So, this team previously reported that a fragment of the extracellular matrix protein, Agrin, promoted cardiac regeneration following myocardial infarction in adult mice. And, in this study the investigators propose to test the therapeutic potential of Agrin in a preclinical porcine model. They performed ischemia reperfusion injuries using balloon occlusion for 60 minutes, followed by either a 3, 7, or 28-day reperfusion period. They demonstrated that local antegrade delivery of recombinant human Agrin, or RH Agrin, to the infarcted pig heart can target the effected regions in an efficient and clinically relevant manner. In fact, a single dose of recombinant human Agrin improved heart function, reduced infarct size, reduced fibrosis and reduced adverse remodeling parameters, 28 days post myocardial infarction. Short-term myocardial infarction experiments, along with complementary mirroring studies, revealed myocardial protection, improved angiogenesis, inflammatory suppression, and cell cycle reentry as aggregation mechanisms of action. So in summary, this team demonstrated that a single dose of Agrin was capable of reducing ischemia reperfusion injury and improving heart function. Demonstrating that Agrin could serve as a therapy for patients with acute MI and potentially heart failure. So, this set the stage for future studies in human subjects. Okay. Well, our next paper is clinical and evaluates exposure to air pollution and particle radioactivity with a risk of ventricular arrhythmias. And, it comes to us from Ms. Ajani Peralta from Harvard University. Now, individuals are exposed to air pollution and ionizing radiation from natural sources through inhalation of particles. So, in this study, the team investigated the association between cardiac arrhythmias and short-term exposures to find particulate matter, PM 2.5, and particle radioactivity. So, ventricular arrhythmogenic events were identified among 176 patients with dual chamber implanted cardio defibrillators in Boston, Massachusetts, between the period of time of September 2006 and June 2010. And, patients were assigned exposures based on their residential addresses. So, what did they find? Well, in this high-risk population, those with these defibrillators, intermediate, 21-day parts per million, 2.5 exposure was associated with higher odds of a ventricular arrhythmia event onset among those with known cardiac disease and indication for ICD implantation. But this was independent of particle radioactivity. So, important information coming to us relating to air pollution and ionizing radiation in relation to ventricular events. Next, let's get back to another informative study from the world of basic science. And, this one involves genomic binding patterns of forkhead box protein 01 and how that is implicated in the development of cardiac hypertrophy. The study comes to us from Walter Koch from Temple University and their co-investigators. So, cardiac hypertrophic growth is mediated by changes in gene expression, as well as changes that underlie the increase in cardiomyocyte size. The former is regulated by ischemia reperfusion or loss, while the latter involves incremental increases in the transcriptional elongation activity of Pol II, that is preassembled at the transcription start site, or TSS. The differential regulation of these two distinct processes, by transcription factors, really hasn't been explored. So, this group sought to investigate the forkhead box protein, and we're going to call it FOX01, which is an insulin sensitive transcription factor that is regulated by hypertrophic stimuli in the heart. To date, however, the scope of its gene regulation is also somewhat uncertain. So, to address this, the investigators performed FOX01 chromatin immunoprecipitation deep sequencing, or ChIP-sequencing, in mouse hearts following seven-day isoproterenol injections, transverse aortic constriction, or vehicle injection by sham surgeries. The investigators found that FOX01 may mediate cardiac hypertrophic growth via regulation of Pol II de novo recruitment and pause release. As the latter represents the majority, or almost 59% of FOX01 bound Pol II regulated genes following pressure overload. So, in conclusion, these findings demonstrate the breadth of transcriptional regulation by FOX01 during cardiac hypertrophy, which is important information that should be valuable for future therapeutic targeting. Moving on from basic science and coming back into the world of clinical science. And, this next paper is from Professor Sripal Bangalore from New York University School of Medicine. And, it involves routine revascularization versus initial medical therapy in those with stable ischemic heart disease. So, coronary arterial revascularization is often performed, as we know, in patients with stable ischemic heart disease. And, these authors conducted a PubMed Embase central search for randomized trials, comparing routine revascularization versus an initial conservative strategy in patients with stable ischemic heart disease. The primary outcome was death, and secondary outcomes included cardiovascular death, myocardial infarction, heart failure, stroke, unstable angina, and freedom from angina. And, the trials were stratified by percent stent use, and by percent statin use, to evaluate the outcomes across all of these trials. So, 14 randomized clinical trials that enrolled 14,877 patients followed up for a weighted mean of 4.5 years with a total of 64,678 patient years of follow-up, were used for the study. Most of the trials enrolled patients with preserved left ventricular systolic function, low symptom burden, and they excluded patients with left main coronary artery disease. So, here are the results, revascularization compared with medical therapy alone, was not associated with a reduced risk of death. The trial sequential analysis showed that the cumulative Z curve crossed the futility boundary indicating firm evidence for lack of a 10% or greater reduction in death. Now, revascularization was associated with a reduced non-procedural MI rate, but also with an increased procedural MI rate with, therefore, no overall difference in myocardial infarction incidents. So, another point in this study is that there was a significant reduction in unstable angina and increase in freedom from angina was observed in those that underwent revascularization. Finally, there were no treatment related differences in the risk of heart failure or stroke. So in conclusion, in patients with stable ischemic heart disease, routine revascularization was not associated with improved survival, but was associated with a lower risk of non-procedural myocardial infarction, and unstable angina with greater freedom from angina at the expense of higher rates of peri procedural myocardial infarction. Now, longer term follow-up of trials is needed to assess whether the reduction in these non-fatal spontaneous events actually improves long-term survival. Well, listeners what else is in the issue? Now, we refer to this as what's in the mailbox? So, we have a research letter from Jianyi Zhang regarding the apical resection prolongs the cell cycle activity and promotes myocardial regeneration, after left ventricular injury, in the neonatal pit. We have another research letter from Roger Foo, assigning distal genomic enhancers to cardiac disease-causing genes. What else is in the issue? There's a nice, On My Mind piece, from Anthony Wierzbicki on phenomics, not genomics, for cardiovascular risk assessment. And, there's a Perspective piece from Dr Dana Gal, regarding considerations for triaging elective cases in children with cardiac disease in a time of crisis. Finally, we have an exchange of letters from Dr Daxin Wang and Dr Prabhakara Nagareddy regarding the previously published article, “Neutrophil Derived S100A8/A9 Amplification of Granulopoieses After Myocardial Infarction.” There's a very nice case series regarding an unusual reversible cause of acute high output heart failure complicated by refractory shock from Dr Matthew Durstenfeld. And then, finally, we have an ECG challenge from Mr. Alejandro Cruz-Utrilla, regarding giant T-wave inversion and dyspnea in the time of this coronavirus pandemic. Well, listeners, what a great issue. And now, we get to look forward to that feature discussion from Dr Allan Sniderman, regarding the benefits of early versus delayed primary prevention by lipid lowering therapy. Well, listeners, now we get to move to our feature discussion today and understand a little bit more about lipid management. And, with us, we have Dr Allan Sniderman from McGill University, and our own associate editor, Dr Anand Rohatgi from University of Texas Southwestern in Dallas. Allan, we're going to start with you. Can you give us a little bit of information pertaining to the background, or the hypothesis? Why did you want to perform this particular study? Dr Allan Sniderman: Let us start from where we are in cardiovascular prevention now, which is the risk model. All of the major guidelines, throughout the world, select candidates for statin prevention based on their risk of a cardiovascular event over the next 10 years. Well, that's been a very positive development, but we need to appreciate what the limitations are. Because, tenure risk is so heavily based on age, what it boils down to is that if you're 60 and over, and a male you're going to be eligible. An increasing proportion of women will be eligible as they're older. But, if you're younger, regardless of your other causal factors, you may well not be eligible. And, the net result of that is, almost 50% of all cardiovascular events occur before 60, 65. So, half of the events are occurring before prevention even kicks in. The second point is that you can't get be at risk until you have disease within your arterial tree. So, what we've done is, we're trying to prevent the disease that's already present. So, although it's been a wonderful step forward, we think it's time to start moving beyond that model, and trying to prevent disease itself, rather than preventing people who've already had disease. So, what we decided to do, which we're shifting focus here from risk to causes. Risk, after all, is a consequence of causes. And, when you look at the pathology of atherosclerosis, with a few exceptions such as FH, up until the age of 30, 35, you don't have the complex lesions that can actually cause clinical events. So, we said, "Okay, we'll start at that time point. And, we'll try and quantitate the benefit of earlier intervention, versus later intervention, at different starting points." So, we based our analysis on NHANES, and we identified the people within the NHANES cohort, who would not have been eligible for prevention, based on the current American guidelines. So, we had three age starting categories, 30 to 39, 40 to 49, and 50 to 59. And, we followed them out for 30 years. Our analysis has the same duration of the follow up, but the same final date. That has to be kept in mind because it limits the total benefit on the early starters. And, we further sub divided the groups based on non-HDL cholesterol, into those who had a level above 160, those who had an intermediate level, and those who had a low level because we'd previously shown that non-HDL identifies a high-risk group over 20 to 30 years. We decided we'd look at two different models of the likelihood of the drug preventing events. One is the standard estimates that you would get from the statin clinical trials. But, if you reduce LDL cholesterol by a milli mol of 40 milligrams percent of your language, you reduce risk by about 22%. And, that's our conservative model. Then we took a model that's more biological and based on Mendelian randomization because, as you know, the Mendelian randomization analysis, the benefit, the reduction and event rate per milligram, per deciliter, lower LDL cholesterol is two or three fold greater than in the statin clinical trials. So, Brian Friends had produced a formula in which you blend those two together, the statin estimate and the Mendelian randomization and it varies depending on how early you start. And, that was a more optimistic model, obviously. But the major point I emphasize is that both models showed that if you start early, you do better. Now, how surprising is that? But the benefit depended on the level of non-HDL cholesterol. So starting at age 30, 35 in somebody who has a normal or low non-HDL cholesterol, doesn't really gain you all that much. Starting with somebody who has a high level, different story. In each of the categories, the conservative model, if you did the full 30-year prevention, you get a third to a half reduction in events. And, with the more optimistic model, you get half to two thirds reduction. The closer you get to where you'd start with the guidelines, you're losing that benefit. Then you say, "Okay, well, how optimistic is the optimistic model?" And, all the optimistic model is saying, "Let's remember how we get disease." If we stop the formation of a new lesion, well, that's perfect prevention. Well, we're doing it, present is trying to stabilize existing lesions. The older we get, the more disease we have, the less new lesion we have, the less potential for the big game in prevention. So stopping new lesion formation, it seems to me is from clinical reasoning, a pretty biologically and clinically coherent way to formulate what your prevention strategy should be. What we think we contributed, what we hope we've contributed with this analysis is saying, "Let's take a real step back, let's look at what we're doing and let's see how can we make a Magnus step forward in prevention?" And, the way we think you can do it is to start moving away from exclusively a risk paradigm, because that made sense. You want to treat people who were at risk. We get away from this 10-year duration and start focusing more on the causes, because when we look at an individual, we can measure the causes. Well, we talked about risks, that's a much slipperier concept, because it's a group. Is everybody in the group the same risk? They're clearly not. So, to try and get this to a much more concrete level, that's what we tried to do. Dr Greg Hundley: Very nice, Allen. And so, Anand, why did you select pushing this paper forward? And, what do you think this means for patients with high cholesterols? They're in their 40s and 50s. Help expand on some of what Allan has, so elegantly, described for us. Anand Rohatgi: First of all, Allan, thank you so much for doing this work and sending this to circulation. We were really excited to see this come across our desks, because at the end of the day, this manuscript, this study by Allan Sniderman and others, has very important public health consequences. And, that's why we were really interested in what they found and what they had to say about this topic. And, really what it comes down to is, like Allan mentioned, the trials for statin just really never addressed what to do about risk in younger individuals and over a longer period of time. And so, it was really these genetic studies, the Mendelian randomization studies, that really strongly showed the cumulative exposure effect of higher cholesterol levels, over time, and the benefits of maintaining really low levels. And so, what I think this paper does is it translates those scientific studies, those genetic studies, into a public health concept that's easily digestible for physicians, for clinicians, for people, for patients and stakeholders about what does this all mean? How can we benefit and when? And, I think it's very clear, the earlier you start, the more benefit you get over the life course. And, honestly, the take home message to me, it seems like for most of adulthood, the being able to maintain low risk and low cholesterol levels is the best path forward and not waiting until you find something wrong. And so, we were very, very excited about seeing that message translated in such an easy manner and compelling manner by Allan's work. Dr Greg Hundley: Very nice, 40 to 49-year old with non-HDL cholesterols greater than 160 milligrams per deciliter, would be expected to reduce their average predicted 30-year risk by 17%. That's just amazing. Allan, very quickly, in a minute, what do you think is the next study that needs to be performed in this area? Dr Allan Sniderman: I don't think we're ever going to get the primary prevention trial that we want, because it's too many people, too early, and we already know too much. I think the thing that we really need to do is figure out how we're going to most accurately measure the cause within the individual. I'll put in a plug for FOV, because I think it is a more effective measure, even the non-HDL cholesterol. And, I think that when we commit people or when we invite people to take medications early in life, we ought to be doing it on the strongest ground possible. I want to say thank you to be review process at Circulation, because the final paper that came out is not the paper that went in. The reviewers made superb, tough criticisms of this paper and not of the calculations, but of the way we were expressing it. And, they forced us to rethink and reimagine the context, in which the final form of this paper appears. So, we have a big thank you to our reviewers. We have a thank you to the patients of the editorial staff at Circulation. And, I have a big thank you to my collaborators, because they're really the strength of the paper. Dr Greg Hundley: Very nice, well listeners, it's been another great week here at Circulation and another very important piece of information in this feature discussion regarding monitoring and evaluating non-HDL cholesterol in the earlier years, and really considering initiation of statin therapy, to prevent the atherosclerotic lesions from forming, not just stabilize them later in life afterwards. Thanks to Anand Rohatgi and also Allan Sniderman for bringing us this wonderful article. We hope you have a great week. This program is copyright the American Heart Association 2020.
In this episode of the Epigenetics Podcast, we caught up with Professor Tom Moss from Université Laval in Québec City, Canada to talk about his work on the chromatin structure and dynamics at ribosomal RNA genes. Dr. Tom Moss has been a member of the Department of Molecular Biology, Medical Biochemistry, and Pathology at the Laval University School of Medicine since he was recruited from the University of Portsmouth in the United Kingdom in 1986. Since then he focused on the ribosomal transcription factor Upstream Binding Factor (UBF) and how it regulates the chromatin structure at ribosomal RNA genes (rDNA). UBF binds to the rDNA as a dimer where it leads to six in-phase bends and induces the formation of the ribosomal enhanceosome. This enhanceosome is required for the initial step in formation of an RNA polymerase I initiation complex, and therefore plays an important role in regulating the expression of ribosomal RNA genes. In this Interview, we discuss the function of UBF on the rDNA, how UBF impacts the chromatin landscape at rRNA genes, the role of DNA methylation in this process, and how UBF influences the structure of the nucleolus. References D. Bachvarov, T. Moss (1991) The RNA polymerase I transcription factor xUBF contains 5 tandemly repeated HMG homology boxes (Nucleic Acids Research) DOI: 10.1093/nar/19.9.2331 V. Y. Stefanovsky, D. P. Bazett-Jones, … T. Moss (1996) The DNA supercoiling architecture induced by the transcription factor xUBF requires three of its five HMG-boxes (Nucleic Acids Research) DOI: 10.1093/nar/24.16.3208 V. Y. Stefanovsky, G. Pelletier, … T. Moss (2001) DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules (Nucleic Acids Research) DOI: 10.1093/nar/29.15.3241 Elaine Sanij, Jeannine Diesch, … Ross D. Hannan (2015) A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes (Genome Research) DOI: 10.1101/gr.176115.114 Tom Moss, Jean-Clement Mars, … Marianne Sabourin-Felix (2019) The chromatin landscape of the ribosomal RNA genes in mouse and human (Chromosome Research) DOI: 10.1007/s10577-018-09603-9 Contact Active Motif on Twitter Epigenetics Podcast on Twitter Active Motif on Linked-In Active Motif on Facebook eMail: podcast@activemotif.com
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06
Gene transcription is a fundamental process of the living cell. Eukaryotic transcription of messenger RNA requires the regulated recruitment of the conserved transcribing enzyme RNA polymerase (Pol) II to the gene promoter. The most heavily regulated step is transcription initiation that involves the ordered assembly of Pol II, the general transcription factors (TF) -IIA, -IIB, -IID, -IIF, -IIE, -IIH and the co-activator Mediator complex. Mediator communicates between transcription regulators and Pol II, and is associated with human disease. Mediator from the yeast Saccharomyces cerevisiae (Sc) has a molecular mass of 1.4 megadaltons and contains 25-subunits that constitute a head, middle, tail and kinase module. The core of Mediator contains the head and middle modules that are essential for viability in Sc, and directly contact Pol II. Mediator co-operates with TFIIH, to assist assembly and stabilization of the transcription initiation complex and stimulate TFIIH kinase activity. Because of the large size and complexity of Mediator and the initiation machinery, the underlying mechanism remains poorly understood. In this work we studied the structure and function of Mediator head and middle modules, the structure of the reconstituted Pol II-core Mediator transcription initiation complex, and reveal mechanisms of transcription regulation. We report the crystal structure of the 6-subunit Schizosaccharomyces pombe Mediator head module at 3.4 Å resolution. The structure resembles the head of a crocodile and reveals eight elements that are part of three domains named neck, fixed jaw and movable jaw. The neck contains a spine, shoulder, arm and finger. The arm and essential shoulder elements contact the remainder of Mediator and Pol II. The head module jaws and central joint, important for transcription, also interact with Mediator and Pol II. The Sp head module structure is conserved and revises a 4.3 Å model of the Sc head module, explains known mutations, and provides an atomic model for one half of core Mediator. We further propose a model of the Mediator middle module based on protein crosslinking and mass spectrometry. To determine how Mediator regulates initiation, we prepared recombinant Sc core Mediator by co-expression of its 15 subunits in bacteria. Core Mediator is active in transcription assays and bound an in vitro reconstituted core initially transcribing complex (cITC) that contains Pol II, the general factors TFIIB, TBP, TFIIF, and promoter DNA. We determined the cryo-electron microscopy structure of the initially transcribing core initiation complex at 7.8 Å resolution. The structure reveals the arrangement of DNA, TBP, TFIIB, and TFIIF on the Pol II surface, the path of the complete DNA template strand and three TFIIF elements. The ‘charged helix’ and ‘arm’ of TFIIF subunit Tfg1, reach into the Pol II cleft and may stabilize open DNA. The linker region of TFIIF subunit Tfg2 extends between Pol II protrusion and TFIIB, and may stabilize TFIIB. The structure agrees with its human counterpart, and suggests a conserved architecture of the core initiation complex. Finally, we determined the cryo-electron microscopy architecture of the cITC-core Mediator complex at 9.7 Å resolution. Core Mediator binds Pol II at the Rpb4/Rbp7 stalk close to the carboxy-terminal domain (CTD). The Mediator head module contacts the Pol II dock and TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module ‘plank’ domain touches the Pol II foot and may control polymerase conformation allosterically. The Med14 subunit bridges head and middle modules with a ‘beam’, and connects to the tail module that binds transcription activators located on upstream DNA. The ‘arm’ and ‘hook’ domains of core Mediator form part of a ‘cradle’ that may position CTD and the TFIIH kinase to stimulate Pol II phosphorylation. Taken together, our results provide a structural framework to unravel the role of Mediator in transcription initiation and determine mechanisms of gene regulation.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06
Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II). This process is tightly regulated and coupled to RNA processing. Many transcription and RNA processing factors are recruited to Pol II via its conserved C-terminal domain (CTD) containing 27 heptapeptide repeats of the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 in Saccharomyces cerevisiae. These repeats can be differentially phosphorylated during the transcription cycle serving as a code for interacting factors. During transcription initiation, Ser5 is phosphorylated at the 5’-end of genes and this phosphorylation is required for RNA capping enzyme binding. During transcription elongation, the Pol II CTD becomes phosphorylated at Tyr1 and Ser2 and binds the elongation factor Spt5. Spt5 also contains a repetitive C-terminal region (CTR) required for cotranscriptional recruitment of proteins. At the 3’-end of genes, Ser2-phosphorylated Pol II associates with the cleavage and polyadenylation factor (CPF) and is dephosphorylated at Tyr1 residues. This work shows that CPF is a Pol II CTD phosphatase and that its subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, Glc7 activity is required for normal Tyr1 dephosphorylation at the polyadenylation (pA) site, for recruitment of termination factors Pcf11 and Rtt103, and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing and transcription termination are coupled via CPF-dependent Pol II Tyr1 dephosphorylation. Additionally, 19 kinases were tested for activity on Tyr1 in yeast by selective inhibition or knock-out in vivo. However, none of the candidates was identified as the Tyr1 kinase. Possibly this enzyme is an atypical kinase not known to be involved in transcription so far. Furthermore, this work reports a new role of the Spt5 CTR in recruitment of RNA 3’-end processing factors. The results show that the Spt5 CTR as well as RNA is required for normal recruitment of the pre-mRNA cleavage factor (CF) I to the 3’-end of yeast genes. Genome-wide ChIP profiling detects occupancy peaks of CFI subunits around 100 base pairs downstream of the pA site of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at Ser2 of the CTD. Consistent with this model, the CTR interacts with CFI in vitro, but is not required for pA site recognition and transcription termination in vivo. In summary, we characterized two new aspects of transcription and RNA processing regulation by two different C-terminal repetitive protein domains. CTD Tyr1 phosphorylation, which is removed by Glc7, regulates termination factor recruitment by masking their binding site, the Spt5 CTR is involved in recruitment of CFI. Both results greatly contribute to a more detailed understanding of the mechanisms involved in transcription termination and RNA 3’-end processing.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06
Eukaryotic gene transcription is highly complex and regulation occurs at multiple stages. RNA Polymerase II (Pol II) is recruited to promoter regions of the DNA to initiate transcription. Shortly after initiation, Pol II exchanges initiation factors for elongation factors. After Pol II passes termination signals, the RNA is cleaved and Pol II eventually released from the DNA template. pre-mRNAs are polyadenylated and exported to the cytosol for translation and ultimately degradation. Mechanisms regulating transcription have been studied extensively, but mechanisms of mRNA degradation are less well understood. To monitor mRNA synthesis and degradation, we developed the comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae Sc cells with the use of Schizosaccharomyces pombe Sp cells as internal standard. We show that Sc mutants can buffer mRNA levels and that impaired transcription causes decreased mRNA synthesis rates compensated by decreased decay rates. Conversely, impairing mRNA degradation causes decreased decay rates, but also decreased synthesis rates. Thus, although separated by the nuclear membrane, transcription and mRNA degradation are coupled. In addition to regulated mRNA synthesis, pervasive transcription can be found throughout the genome, governed by an intrinsic affinity of Pol II for DNA. These divergent noncoding RNAs (ncRNAs) stem to a large extent from bidirectional promoters. However, global mechanisms for the termination of ncRNA synthesis that could act as a transcriptome surveillance mechanism are not known. It is also unclear if such a surveillance system protects the transcriptome from deregulation. Here we show that ncRNA transcription in Sc is globally restricted by early termination which relies on the essential RNA-binding factor Nrd1. Depletion from the nucleus results in Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) throughout the genome and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at essentially all promoters and antisense NUTs in most 3’-regions of genes. Nrd1 preferentially binds RNA motifs which are enriched in ncRNAs and depleted in mRNAs except in some mRNAs whose synthesis is controlled by transcription attenuation. These results describe a mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and prevent transcriptome deregulation
Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 04/06
So far, much attention has been paid to regulation of transcription. However, it has been realized that controlled mRNA decay is an equally important process. To understand the contributions of mRNA synthesis and mRNA degradation to gene regulation, we developed Dynamic Transcriptome Analysis (DTA). DTA allows to monitor these contributions for both processes and for all mRNAs in the cell without perturbation of the cellular system. DTA works by non-perturbing metabolic RNA labeling that supersedes conventional methods for mRNA turnover analysis. It is accomplished with dynamic kinetic modeling to derive the gene-specific synthesis and decay parameters. DTA reveals that most mRNA synthesis rates result in several transcripts per cell and cell cycle, and most mRNA half-lives range around a median of 11 min. DTA can monitor the cellular response to osmotic stress with higher sensitivity and temporal resolution than standard transcriptomics. In contrast to monotonically increasing total mRNA levels, DTA reveals three phases of the stress response. In the initial shock phase, mRNA synthesis and decay rates decrease globally, resulting in mRNA storage. During the subsequent induction phase, both rates increase for a subset of genes, resulting in production and rapid removal of stress-responsive mRNAs. In the following recovery phase, decay rates are largely restored, whereas synthesis rates remain altered, apparently enabling growth at high salt concentration. Stress-induced changes in mRNA synthesis rates are predicted from gene occupancy with RNA polymerase II. Thus, DTA realistically monitors the dynamics in mRNA metabolism that underlie gene regulatory systems. One of the technical obstacles of standard transcriptomics is the unknown normalization factor between samples, i.e. wild-type and mutant cells. Variations in RNA extraction efficiencies, amplification steps and scanner calibration introduce differences in the global intensity levels. The required normalization limits the precision of DTA. We have extended DTA to comparative DTA (cDTA), to eliminate this obstacle. cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae (Sc) cells with the use of Schizosaccharomyces pombe (Sp) as an internal standard. It therefore allows for direct comparison of RNA synthesis and decay rates between samples. cDTA reveals that Sc and Sp transcripts that encode orthologous proteins have similar synthesis rates, whereas decay rates are five fold lower in Sp, resulting in similar mRNA concentrations despite the larger Sp cell volume. cDTA of Sc mutants reveals that a eukaryote can buffer mRNA levels. Impairing transcription with a point mutation in RNA polymerase (Pol) II causes decreased mRNA synthesis rates as expected, but also decreased decay rates. Impairing mRNA degradation by deleting deadenylase subunits of the Ccr4–Not complex causes decreased decay rates as expected, but also decreased synthesis rates. In this thesis, we provide a novel tool to estimate RNA synthesis and decay rates: a quantitative dynamic model to describe mRNA metabolism in growing cells to complement the biochemical protocol of DTA/cDTA. It can be applied to reveal rate changes for all kinds of perturbations, e.g. in knock-out or point mutation strains, in responses to stress stimuli or in small molecule interfering assays like treatments with miRNA or siRNA inhibitors. In doing so, we show that DTA is a valuable tool for miRNA target validation. The DTA/cDTA approach is in principle applicable to virtually every organism. The bioinformatic workflow of DTA/cDTA is implemented in the open source R/Bioconductor package DTA.
Shona Murphy Sir WILLIAM DUNN School of Pathology, Molecular Biology, Oxford, UK, speaks on "Human small nuclear (sn)RNA genes and the pol II CTD code". This seminar has been recorded by ICGEB Trieste
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 04/06
Eukaryotic nuclear transcription is carried out by three different Polymerases (Pol), Pol I, Pol II and Pol III. Among these, Pol I is dedicated to transcription of the rRNA, which is the first step of ribosome biogenesis, and cell growth is regulated during Pol I transcription initiation by the conserved factor Rrn3/TIF-IA in yeast/human. A wealth of structural information is available on Pol II and its general transcription factors (GTFs). Recently, also the architectures of Pol I and Pol III have been described by electron microscopy and the additional subunits that are specific to Pol I and Pol III have been identified as orthologs of the Pol II transcription factors TFIIF and TFIIE. Nevertheless, we still lack information about the architecture of the Pol I initiation complex and structural data is missing explaining the regulation of Pol I initiation mediated by its central transcription initiation factor Rrn3. The Rrn3 structure solved in this study reveals a unique HEAT repeat fold and indicates dimerization of Rrn3 in solution. However, the Rrn3-dimer is disrupted upon Pol I binding. The Rrn3 structure further displays a surface serine patch. Phosphorylation of this patch represses human Pol I transcription (Mayer et al, 2005; Mayer et al, 2004), and a phospho-mimetic patch mutation prevents Rrn3 binding to Pol I in vitro, and reduces S. cerevisiae cell growth and Pol I gene occupancy in vivo. This demonstrates a conserved regulation mechanism of the Pol I-Rrn3 interaction. Crosslinking indicates that Rrn3 does not only interact with Pol I subunits A43/14, but the interface further extends past the RNA exit tunnel and dock domain to AC40/19. The corresponding region of Pol II binds the Mediator head (Soutourina et al., 2011) that co-operates with TFIIB (Baek et al, 2006). Consistent with this, the Rrn3 binding partner, core factor subunit Rrn7, is predicted to be a TFIIB homologue. Taken together, our results provide the molecular basis of Rrn3-regulated Pol I initiation and cell growth and indicate a universally conserved architecture of eukaryotic transcription initiation complexes.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 03/06
Mediator is a central coactivator complex required for regulated transcription by RNA polymerase (Pol) II in all eukaryotes. Budding yeast Mediator has a size of 1.4 MDa and consists of 25 subunits arranged in the head, middle, tail, and kinase modules. It is thought that Mediator forms an interface between the general RNA polymerase (RNA Pol) II machinery and transcriptional activators leading to promotion of pre-initiation complex (PIC) assembly. Mediator middle module from budding yeast consists of seven subunits Med1, 4, 7, 9, 10, 21, and 31 and was investigated during this thesis both structurally and functionally. Previously, the structure of a subcomplex comprising the C-terminal region of Med7 (Med7C) and Med21 was solved by X-ray crystallography and protocols for obtaining larger recombinant complexes were established in the laboratory. As structural and functional studies of Mediator are limited by the availability of protocols for the preparation of modules, I pursued these studies and established protocols for obtaining pure endogenous and recombinant complete Mediator middle module. Another subcomplex of the middle module, comprising the N-terminal part of subunit Med7 (Med7N) and the highly conserved subunit Med31 (Soh1) was successfully crystallized and its structure solved during this work. It is found, that it contains a unique structure and acts also as a functional entity (termed submodule). The Med7N/31 submodule shows a novel fold, with two conserved proline-rich stretches in Med7N wrapping around the righthanded four-helix bundle of Med31. In vitro, Med7N/31 is required for activated transcription and can act in trans when added exogenously. In vivo, Med7N/31 has a predominantly positive function on the expression of a specific subset of genes, including genes involved in methionine metabolism and iron transport. Comparative phenotyping and transcriptome profiling identified specific and overlapping functions of different Mediator submodules. Crystallization screening of larger middle module (sub-)complexes did not result in crystal formation, even after removal of some flexible regions. Thus alternative methods were applied to characterize the middle module topology. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion mobility mass spectrometry, limited proteolysis, light scattering, and small angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module, giving a potential explanation of why crystallization of larger complexes was unsuccessful. Moreover, based on systematic protein-protein interaction analysis, a new model for the subunit-subunit interaction network within the middle module of the Mediator is proposed. In this model, the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes Med7N/21, Med7C/31, and Med4/9. The subunits Med1 and Med10, which bridge to the Mediator tail module, bind to both Med7 and Med4. Furthermore, first steps in establishing an in vitro assay to test endogenous and recombinant middle module functionality have been initiated and will provide the basis for future studies.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 03/06
RNA polymerase II (Pol II) is the eukaryotic enzyme responsible for transcribing all protein-coding genes into messenger RNA (mRNA). This thesis describes studies on the molecular mechanisms of Pol II translocation, alpha-amanitin inhibition and DNA lesion recognition by Pol II. To study how Pol II translocates after nucleotide incorporation, we prepared elongation complex (EC) crystals in which pre- and post-translocation states interconvert. Crystal soaking with the inhibitor alpha-amanitin locked the EC in a new state that we identified as a translocation intermediate at 3.4 Å resolution. The DNA base entering the active site occupies a “pre-templating” position above the central bridge helix, which is shifted and occludes the standard templating position. A leucine residue in the trigger loop forms a wedge next to the shifted bridge helix, but moves by 13 Å to close the active site for nucleotide incorporation. Our results support a Brownian ratchet mechanism of elongation that involves swinging of the trigger loop between open, wedged, and closed positions, and suggest that alpha-amanitin impairs nucleotide incorporation and translocation by trapping the trigger loop and bridge helix in a translocation intermediate. Cells use transcription-coupled repair (TCR) to efficiently eliminate DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs). Here we present the structure-based mechanism for the first step in eukaryotic TCR, CPD-induced stalling of Pol II. A CPD in the transcribed strand slowly passes a translocation barrier, and enters the polymerase active site. The CPD 5’-thymine then directs uridine monophosphate (UMP) misincorporation into mRNA, which blocks translocation. Artificial replacement of the UMP by adenosine monophosphate (AMP) enables CPD bypass, thus Pol II stalling requires CPD-directed misincorporation. In the stalled complex, the lesion is inaccessible, and the polymerase conformation is unchanged. This is consistent with non-allosteric recruitment of repair factors and excision of a lesion-containing DNA fragment in the presence of Pol II. CPD recognition is compared with the recognition of a cisplatin-induced guanine-guanine intrastrand crosslink. Similarities and differences in the detailed mechanism of transcriptional stalling at the two different dinucleotide lesions are discussed.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
Synthesis of ribosomal RNA by RNA polymerase (Pol) I is the first step in ribosome biogenesis and a regulatory switch in eukaryotic cell growth. In this thesis a reproducible large-scale purification protocol for Pol I from S. cerevisiae could be developed. Crystals were obtained, diffraction to < 4 Å could be recorded, however, the enormously complex non-crystallographic symmetry impeded structure solution. Switching to cryo-electron microscopy, the structure of the complete 14-subunit enzyme could be solved to 12 Å resolution, a homology model for the core enzyme could be generated, and the crystal structure of the subcomplex A14/43 could be solved. In the resulting hybrid structure of Pol I, A14/43, the clamp, and the dock domain contribute to a unique surface interacting with promoter-specific initiation factors. The Pol I-specific subunits A49 and A34.5 form a heterodimer near the enzyme funnel that acts as a built-in elongation factor, and is related to the Pol II-associated factor TFIIF. In contrast to Pol II, Pol I has a strong intrinsic 3’-RNA cleavage activity, which requires the C-terminal domain of subunit A12.2, and apparently enables rRNA proofreading and 3’-end trimming.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
TFIIF is the only general transcription factor that has been implicated in the preinitiation complex assembly, open complex formation, initiation and transcription elongation. In addition, TFIIF stimulates Fcp1, a central phosphatase needed for recycling of RNA polymerase II (Pol II) after transcription by dephosphorylation of the Pol II C-terminal domain (CTD). This thesis reports the X-ray structure of the small CTD phosphatase Scp1, which is homologous to the Fcp1 catalytic domain. The structure shows a core fold and an active center similar to phosphotransferases and –hydrolases that solely share a DXDX(V/T) signature motif with Fcp1/Scp1. It was further demonstrated that the first aspartate in the signature motif undergoes metalassisted phosphorylation during catalysis, resulting in a phosphoaspartate intermediate that was structurally mimicked with the inhibitor beryllofluoride. Specificity may result from CTD binding to a conserved hydrophobic pocket between the active site and an insertion domain that is unique to Fcp1/Scp1. Fcp1 specificity may additionally arise from phosphatase recruitment near the CTD via the Pol II subcomplex Rpb4/7, which is shown to be required for Fcp1 binding to the polymerase in vitro. Until now, the main impediment in the high resolution crystallographic studies of TFIIF in complex with Pol II and other members of transcription machinery was unavailability of soluble, stoichiometric TFIIF complex in sufficient amounts. This thesis reports on the development of the overexpression system in yeast and a purification protocol that enabled for the first time to isolate milligram amounts of a pure and soluble, 15-subunit (~0,7 MDa) stoichiometric Pol IITFIIF complex. Such complex together with the promoter DNA, RNA, TBP and TFIIB assembles in vitro into the yeast initially transcribing complex, which can now be studied structurally.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
The Mediator of transcriptional regulation is the central coactivator that enables a response of RNA polymerase II (Pol II) to activators and repressors. Yeast Mediator has a size of more than one MDa and consists of 25 different polypeptides. Biochemical studies defined three Mediator modules in yeast, the head (MED17) the middle (MED9/MED10) and the tail (MED15) modules. During this work, an E.coli coexpression-copurification system was developed, which allowed to study pairwise interactions of Mediator middle module subunits. With the help of this system I reconstituted a complex of two essential and conserved yeast Mediator middle module proteins, the MED7/MED21 heterodimer, and solved its crystal structure. The heterodimer forms an extended structure, which spans one third of the Mediator length, and almost the diameter of Pol II. It shows a four helix bundle and a coiled-coil protrusion connected by a flexible hinge. Multiple conserved patches can be identified on the surface, which allow for assembly of the middle module. A combination of the coexpression-copurification system and assembly of subcomplexes allowed the reconstitution of a five-subunit Mediator middle module subcomplex. The reconstituted subcomplex is able to bind Pol II in vitro. MED6 associates with the middle module and forms a bridge to the head module. The potential flexibility of this bridge and the MED7/MED21 hinge can account for changes in Mediator structure upon its binding to Pol II or to activators.
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
RNA polymerase II (Pol II) is the central enzyme, that synthetizes all mRNA in eukaryotic cells. In this work, I solved the structure of the complete, initiation-competent 12-subunit yeast RNA polymerase II at 3.8 Å. I also solved the structure of the Pol II subcomplex of Rpb4/7 alone at 2.3 Å resolution. These structures reveal the details of Pol II assembly from 12 subunits and give important insights into the initiation of transcription. The refined, atomic model of the complete 12-subunit Pol II enabled homology modeling of the two other nuclear RNA polymerases. In Pol I and Pol III, 65 % and 77 % of the Pol II fold are conserved, respectively. Together with a recent structure of a Pol II elongation complex, these results show that the basic mechanism of transcription applies also to the two other nuclear RNA polymerases
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
This thesis describes crystal structures of complete, 12-subunit yeast RNA polymerase II (Pol II) in complex with a synthetic transcription bubble and product RNA, with an NTP substrate analogue, and in complex with the transcription elongation factor TFIIS. The structure of the Pol II-transcription bubble-RNA complex reveals incoming template and non-template DNA, a seven base-pair DNA-RNA hybrid, and three nucleotides each of separating DNA and RNA. Based on this structure, those parts of Pol II were identified which are involved in separating template DNA from non-template DNA before the active site, and DNA from product RNA at the upstream end of the DNA-RNA hybrid. In both instances, strand separation can be explained by Pol II-induced duplex distortions. Only parts of the complete transcription bubble present in the complexes are ordered in the crystal structure, explaining the way in which high processivity of Pol II is reconciled with rapid translocation along the DNA template. The presence of an NTP substrate analogue in a conserved putative pre-insertion site was unveiled in a Pol II-transcription bubble-RNA complex crystal soaked with the substrate analogue GMPCPP. The structure of the Pol II-TFIIS complex was obtained from Pol II crystals soaked with TFIIS. TFIIS extends from the Pol II surface to the active site and complements the active site with two essential and invariant acidic residues for hydrolytic RNA cleavage. TFIIS also induces extensive structural changes in Pol II that reposition nucleic acids, in particular RNA, near the active centre. These results support the idea that Pol II contains a single tuneable active site for RNA polymerisation and cleavage. The technical obstacles imposed by crystal structure determination of large, transient protein-DNA-RNA complexes were overcome by two novel, fluorescence-based assays to monitor and optimise the composition of the crystals. Both assays are not limited to Pol II complexes, but can serve as a general tool for the crystallographic community.