DOCSLIB.ORG

Transcriptional Arrest of Yeast RNA Polymerase II by Escherichia Coli

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Transcriptional Arrest of Yeast RNA Polymerase II by Escherichia Coli Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6606-6610, July 1993 Biochemistry Transcriptional arrest of yeast RNA polymerase II by Escherichia coli rho protein in vitro (transcription termination/RNA 3' ends/RNA processing) SHWU-YUAN WU AND TERRY PLATT* Department of Biochemistry, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642 Communicated by Michael J. Chamberlin, April 20, 1993 (receivedfor review May lS, 1992) ABSTRACT A promoter-independent assay utilizing initiation (3). Premature termination (attenuation) in the poly(dC)-tailed DNA templates has revealed that Saccharomy- 5'-proximal regions of several genes in mammalian systems ces cerevisiae whole-cell extracts can be proficient for tran- has been examined by either promoter-directed transcription scription by the endogenous yeast RNA polymerase H as well in cell extract systems (4-10) or promoterless transcription as for correct 3'-end RNA processing. Our attempts to examine with purified pol II (11-14). These sites may be intrinsic the fate of polymerase II itself were inconclusive, because only terminators for pol II and contain distinct signals such as trace btanscription products corresponded to the expected size RNA stem-loop structures (5, 7, 14), DNA bending in T-rich of terminated RNA species. Transcription in our processing- tracts (13), or other, structureless sequences (6). proficient extract was thus insufficient to cause termination. To While these examples illustrate an important role for test our system with a known, albeit heterologous, signal, we transcription attenuation in the regulation of eukaryotic gene examined a dC-tailed template carrying the E. col rho- expression, the fate of pol II in 3' noncoding regions after it dependent termination signal £p t' in the yeast extract. Tran- passes the poly(A) site(s) and terminates at downstream scripts from this template were not susceptible to processing, sequences is obscure, both temporally and mechanistically but addition of rho protein resulted in two distinct truncated (15). In higher eukaryotes, mutation studies and nuclear transcripts that could not be chased by excess unlabeled run-on experiments have suggested that the RNA processing nucleotides. These RNA species thus represented stably paused events associated with mature 3'-end formation may them- or terminated polymerase II products, and their absence when selves be involved in regulating the efficiency oftranscription a mutated unresponsive tip t' template was used affirmed that termination, despite the progress of pol II for long distances they were due to the effects of rho. E. col RNA polymerase past the poly(A) site (16-18). was also In Saccharomyces cerevisiae, RNA processing events also added to a yeast extract pretreated with a-amanitin generate the mature 3' ends of mRNA transcripts (19, 20). halted by rho at these same two sites. A mutated rho protein, Unlike the situation in higher eukaryotes, considerable cir- while only partly defective with E. coli polymerase, failed to cumstantial evidence indicates that yeast pol II does not provoke arrest when transcription was carried out by RNA proceed far beyond the polyadenylylation site before it polymerase H. Thus, functional rho and its cognate site, tip t', terminates transcription (21-23). To identify the sites and the appear necessary and sufficient to elicit the production of signals specifying transcription termination for pol II, we truncated transcripts by RNA polymerase II in a yeast whole- have developed a coupled system containing the 3' RNA cell extract. The ability of rho to halt the eukaryotic enzyme processing and pol II activities. In such a system, utilizing strengthens the likelihood that a rho-like helicase may be dC-tailed templates to achieve promoter-independent initia- involved in RNA polymerase H transcription termination. tion by pol II, no substantial level oftranscription termination is detectable under conditions either allowing or blocking the Transcription termination is required to complete RNA syn- processing events. However, we show that on a template thesis and prevent transcriptional interference with down- carrying the bacterial rho-dependent termination site trp t', stream genes. It occurs when the RNA polymerase dissoci- E. coli termination factor rho can halt RNA elongation by the ates from the DNA template and the nascent RNA is re- yeast transcription complex, suggesting the possible exis- leased. Termination mechanisms are classified into two tence of rho-like termination factors in yeast and other major categories (1). Intrinsic termination occurs as RNA eukaryotic organisms. polymerase stops in direct response to signals encoded in the RNA transcript and/or the DNA template. Alternative mech- anisms require the action of some additional trans-acting MATERIALS AND METHODS factor(s) to terminate RNA elogation by the polymerase DNA Templates with Deoxycytidylylated 3' Ends. Fifty mi- molecules. Among identified bacterial termination factors, crograms of CsCl-purified DNA was linearized, ethanol- the involvement of Escherichia coli rho protein in transcrip- precipitated, and dissolved in 50 MI of 0.2 M potassium tion termination is best understood (2). cacodylate, pH 7.2/1 mM CoCl2/2 mM 2-mercaptoethanol/ In eukaryotes, the study of RNA polymerase II (pol II) 0.2 mM dCTP with 60 units ofterminal deoxynucleotidyltrans- termination is complicated by 3'-end RNA processing, which ferase (Ratliff Biochemicals, Los Alamos, NM). The mixture produces mature mRNA 3' ends via specific endonucleolytic was then incubated at 37°C for 40 min, followed by ethanol cleavage and polyadenylylation. To distinguish between ter- precipitation and wash. After digestion by the second restric- mination and RNA processing events, nuclear run-on studies tion enzyme, the DNA templates specifically poly(dC)-tailed in vivo or transcription experiments in vitro are required. at one 3' end were purified from agarose gels and dissolved in However, the former are tedious and time-consuming, and 50 ,ul of doubly distilled water. The lengths of the poly(dC)- the latter are difficult due to the complexities of pol II tails averaged 100 nt, estimated by comparisons with a DNA size marker of 123 nt (Bethesda Research Laboratories). The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: pol II, RNA polymerase II. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6606 Downloaded by guest on September 27, 2021 Biochemistry: Wu and Platt Proc. Natl. Acad. Sci. USA 90 (1993) 6607 Transcription in Vitro. Yeast whole-cell extracts were RESULTS prepared as described (20), except that we used only 180 mg Transcription in Vitro by Endogenous pol II in a Processing- ofammonium sulfate per ml in the fractionation step, instead Proficient Extract. To avoid the complications of promoter of220 mg. Ten microliters oftranscription mixture contained initiation for yeast pol II transcription in vitro, we took 50-100 ng of poly(dC)-tailed DNA template, 0.5-2 ,ul of advantage of promoter-independent transcription protocols extract (20 mg ofprotein per ml), 8 mM Hepes/KOH (pH 7.0), utilizing linear DNA templates extended at one 3' end by a 50 mM potassium acetate, 5 mM magnesium acetate, 1 mM poly(dC) tail (25, 26). We prepared 3' dC-extended CYCI dithiothreitol, three nonradioactive NTPs at 500 ,uM, and S DNAs (Fig. 1A) to test transcription in yeast whole-cell ,uCi of [a-32P]CTP or -UTP (800 Ci/mmol, NEN; 1 Ci = 37 extracts, which are proficient in 3' processing of many yeast GBq) brought to the desired concentration with unlabeled transcripts, including ones corresponding to the CYCI gene nucleotide. Incubation was at 30°C for 20 min unless other- (19, 20). There was sufficient endogenous RNA polymerase wise specified. In the chase experiments using yeast CYCI as activity to support transcription (Fig. 1B, lane 1), and its templates, the reaction was carried out with 20 ,uM inhibition by a-amanitin (10 pg/ml) (lane 2) indicates that it [a-32P]CTP for 10 min and then with 500 ,uM unlabeled CTP was due to pol II. for another 10 min. For rho-dependent termination, a 5-min Since plasmid stability assays have suggested that a tran- preincubation of DNA template and RNA polymerase was scriptional block occurs within 100 nt beyond the CYCI followed by the addition ofwild-type or mutant rho (replaced poly(A) site (23), we prepared three related poly(dC)-tailed by bovine serum albumin in reaction mixtures lacking rho) CYCI templates. Each ofthese carried 470 nt of3' noncoding and nucleotide mix (10 ,uM [a-32P]UTP/2.5 mM ATP/500 ,uM sequence, with end extensions from either the coding region GTP/500 ,uM CTP). After the reactions were incubated at or vector sequences (Fig. 1A). Templates 1 and 2 were 30°C for 3 min, a chase was carried out with 500 ,uM identical in their 5' regions, but the former had an additional nonradioactive UTP for 10 min. For pretreatment with 130 nt of vector sequence at the 3' terminus. Template 2 and a-amanitin, 2 p.l ofextract and 1 ,ul ofa-amanitin solution (100 3 contained the same 3' regions, but the latter had an extra 90 5 nt (of the coding sequence) in its 5' portion. When these pg/ml) were incubated on ice for min and then used for templates were incubated in extracts under transcription rho-dependent termination as described above. Wild-type E. conditions (see Materials and Methods), we detected runoff coli rho factor was purified from cells carrying the overpro- and processed transcripts in each case with the sizes pre- ducing vector p39ASE (24). Reactions were stopped and the dicted by processing at the major in vivo polyadenylylation mixtures were phenol/chloroform-extracted before ethanol site (28) (Fig. 1B, lanes 3-6). The consistency in the lengths precipitation (20).
Load more

Recommended publications
  • Rho Dependent Termination of Transcription in Prokaryotes
    Rho Dependent Termination Of Transcription In Prokaryotes Organoleptic Tymon scrimshaws no athenaeums yean fleetly after Teodorico whiled tunably, quite scrawliest. Amalgamative and honeyed Ximenes syllables her bullheads picket stork's-bill and debouch visually. Colory Jeffery concentred her avidity so injudiciously that Sergio briquet very injunctively. Dna in prokaryotic polymerase in termination may vary. Dna sequence by enzymes and fall off of transcripts, and marylène bertrand for the conformation of rho in the stalled rna helicases, rho dependent termination of in transcription. The other physiological functions are the basis for rna polymerase and longer as elongation complex forming a series of one of rho termination in transcription factors recognizing promoters in four steps. Eukaryotes require cookies from prokaryotes, depending on a fifth subunit involved. Thus, when translation termination occurs within same gene it the cause transcriptional termination, preventing expression of downstream genes. Atp synthase alpha and therefore be specialized rnas make a bsr, in termination of rho transcription prokaryotes. Dna must accept cookies and also found later in part because of rho termination occurs by the initiation site that bind to understand how they cannot be logged in replicating dna. Concerned about the Coronavirus? It is made rna polymerase causes rna in termination of rho transcription termination mechanisms to specific genes. Depending upon request your changes indicating that prokaryotic rnap with and prokaryotes. The DNA strand within this an is transcribed by the RNA polymerase. The basic promoter region in prokaryotic transcription is referred to two the Pribnow box. Formation of which prokaryotic and on the hfq is shown as the transcript of action of rho dependent termination transcription in prokaryotes have far more about ten base that elongation complex cell components and cell.
    [Show full text]
  • Resistance to Rifampicin: a Review
    The Journal of Antibiotics (2014) 67, 625–630 & 2014 Japan Antibiotics Research Association All rights reserved 0021-8820/14 www.nature.com/ja REVIEW ARTICLE Resistance to rifampicin: a review Beth P Goldstein Resistance to rifampicin (RIF) is a broad subject covering not just the mechanism of clinical resistance, nearly always due to a genetic change in the b subunit of bacterial RNA polymerase (RNAP), but also how studies of resistant polymerases have helped us understand the structure of the enzyme, the intricacies of the transcription process and its role in complex physiological pathways. This review can only scratch the surface of these phenomena. The identification, in strains of Escherichia coli, of the positions within b of the mutations determining resistance is discussed in some detail, as are mutations in organisms that are therapeutic targets of RIF, in particular Mycobacterium tuberculosis. Interestingly, changes in the same three codons of the consensus sequence occur repeatedly in unrelated RIF-resistant (RIFr) clinical isolates of several different bacterial species, and a single mutation predominates in mycobacteria. The utilization of our knowledge of these mutations to develop rapid screening tests for detecting resistance is briefly discussed. Cross-resistance among rifamycins has been a topic of controversy; current thinking is that there is no difference in the susceptibility of RNAP mutants to RIF, rifapentine and rifabutin. Also summarized are intrinsic RIF resistance and other resistance mechanisms. The Journal of Antibiotics (2014) 67, 625–630; doi:10.1038/ja.2014.107; published online 13 August 2014 INTRODUCTION laboratory studies and emerging in patients who received mono- In celebrating the life of Professor Piero Sensi and his discovery of therapy with RIF.
    [Show full text]
  • Bicyclomycin Sensitivity and Resistance Affect Rho Factor-Mediated Transcription Termination in the Tna Operon of Escherichia Coli
    JOURNAL OF BACTERIOLOGY, Aug. 1995, p. 4451–4456 Vol. 177, No. 15 0021-9193/95/$04.0010 Copyright 1995, American Society for Microbiology Bicyclomycin Sensitivity and Resistance Affect Rho Factor-Mediated Transcription Termination in the tna Operon of Escherichia coli CHARLES YANOFSKY* AND VIRGINIA HORN Department of Biological Sciences, Stanford University, Stanford, California 94305-5020 Received 13 March 1995/Accepted 27 May 1995 The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be acquired by mutations at any one of three loci, two of which appear to be rho and rpoB. Zwiefka et al. (24) found that the antibiotic bicyclomycin segment and interacts with the transcribing RNA polymerase (bicozamycin), an inhibitor of the growth of several gram- molecule, causing it to terminate transcription (7, 9).
    [Show full text]
  • Termination of RNA Polymerase II Transcription by the 5’-3’ Exonuclease Xrn2
    TERMINATION OF RNA POLYMERASE II TRANSCRIPTION BY THE 5’-3’ EXONUCLEASE XRN2 by MICHAEL ANDRES CORTAZAR OSORIO B.S., Universidad del Valle – Colombia, 2011 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2018 This thesis for the Doctor of Philosophy degree by Michael Andrés Cortázar Osorio has been approved for the Molecular Biology Program by Mair Churchill, Chair Richard Davis Jay Hesselberth Thomas Blumenthal James Goodrich David Bentley, Advisor Date: Aug 17, 2018 ii Cortázar Osorio, Michael Andrés (Ph.D., Molecular Biology) Termination of RNA polymerase II transcription by the 5’-3’ exonuclease Xrn2 Thesis directed by Professor David L. Bentley ABSTRACT Termination of transcription occurs when RNA polymerase (pol) II dissociates from the DNA template and releases a newly-made mRNA molecule. Interestingly, an active debate fueled by conflicting reports over the last three decades is still open on which of the two main models of termination of RNA polymerase II transcription does in fact operate at 3’ ends of genes. The torpedo model indicates that the 5’-3’ exonuclease Xrn2 targets the nascent transcript for degradation after cleavage at the polyA site and chases pol II for termination. In contrast, the allosteric model asserts that transcription through the polyA signal induces a conformational change of the elongation complex and converts it into a termination-competent complex. In this thesis, I propose a unified allosteric-torpedo mechanism. Consistent with a polyA site-dependent conformational change of the elongation complex, I found that pol II transitions at the polyA site into a mode of slow transcription elongation that is accompanied by loss of Spt5 phosphorylation in the elongation complex.
    [Show full text]
  • 3-End Formation of Baculovirus Late Rnas
    JOURNAL OF VIROLOGY, Oct. 2000, p. 8930–8937 Vol. 74, No. 19 0022-538X/00/$04.00ϩ0 Copyright © 2000, American Society for Microbiology. All Rights Reserved. 3Ј-End Formation of Baculovirus Late RNAs 1 1,2 JIANPING JIN AND LINDA A. GUARINO * Departments of Biochemistry and Biophysics1 and Entomology,2 Texas A&M University, College Station, Texas 77843-2128 Received 13 March 2000/Accepted 30 June 2000 Baculovirus late RNAs are transcribed by a four-subunit RNA polymerase that is virus encoded. The late viral mRNAs are capped and polyadenylated, and we have previously shown that capping is mediated by the LEF-4 subunit of baculovirus RNA polymerase. Here we report studies undertaken to determine the mecha- Downloaded from nism of 3؅-end formation. A globin cleavage/polyadenylation signal, which was previously shown to direct 3؅-end formation of viral RNAs in vivo, was cloned into a baculovirus transcription template. In vitro assays with purified baculovirus RNA polymerase revealed that 3؅ ends were formed not by a cleavage mechanism but rather by termination after transcription of a T-rich region of the globin sequence. Terminated RNAs were released from ternary complexes and were subsequently polyadenylated. Mutational analyses indicated that the T-rich sequence was essential for termination and polyadenylation, but the poly(A) signal and the GT-rich region of the globin polyadenylation/cleavage signal were not required. Termination was not dependent on ATP hydrolysis, indicating a slippage mechanism. http://jvi.asm.org/ mRNA 3Ј-end formation is a complicated process that re- promoters used for overexpression in baculovirus vectors be- quires protein-nucleic acid and protein-protein interactions.
    [Show full text]
  • Regulatory Interplay Between Small Rnas and Transcription Termination Factor Rho Lionello Bossi, Nara Figueroa-Bossi, Philippe Bouloc, Marc Boudvillain
    Regulatory interplay between small RNAs and transcription termination factor Rho Lionello Bossi, Nara Figueroa-Bossi, Philippe Bouloc, Marc Boudvillain To cite this version: Lionello Bossi, Nara Figueroa-Bossi, Philippe Bouloc, Marc Boudvillain. Regulatory interplay be- tween small RNAs and transcription termination factor Rho. Biochimica et Biophysica Acta - Gene Regulatory Mechanisms , Elsevier, 2020, pp.194546. 10.1016/j.bbagrm.2020.194546. hal-02533337 HAL Id: hal-02533337 https://hal.archives-ouvertes.fr/hal-02533337 Submitted on 6 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Regulatory interplay between small RNAs and transcription termination factor Rho Lionello Bossia*, Nara Figueroa-Bossia, Philippe Bouloca and Marc Boudvillainb a Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198, Gif-sur-Yvette, France b Centre de Biophysique Moléculaire, CNRS UPR4301, rue Charles Sadron, 45071 Orléans cedex 2, France * Corresponding author: [email protected] Highlights Repression
    [Show full text]
  • Sral Srna Interaction Regulates the Terminator by Preventing Premature Transcription Termination of Rho Mrna
    SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA Inês Jesus Silvaa,1, Susana Barahonaa,2, Alex Eyraudb,2, David Lalaounab, Nara Figueroa-Bossic, Eric Masséb, and Cecília Maria Arraianoa,1 aInstituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, 2780-157 Oeiras, Portugal; bRNA Group, Department of Biochemistry, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada; and cInstitute for Integrative Biology of the Cell (I2BC), Commissariat à l’énergie atomique, CNRS, Université Paris-Sud, Université Paris-Saclay, 91198 Gif-sur-Yvette, France Edited by Tina M. Henkin, The Ohio State University, Columbus, OH, and approved December 28, 2018 (received for review July 5, 2018) Transcription termination is a critical step in the control of gene sequence, leading to changes in translation and/or mRNA degra- expression. One of the major termination mechanisms is mediated dation (9–11). However, distinct mechanisms of action have been by Rho factor that dissociates the complex mRNA-DNA-RNA poly- increasingly reported in the literature (11). For instance, the Sal- merase upon binding with RNA polymerase. Rho promotes termina- monella sRNA ChiX was shown to induce premature transcription tion at the end of operons, but it can also terminate transcription termination within the coding sequence of chiP as a result of its within leader regions, performing regulatory functions and avoiding interaction with 5′-UTR of the operon chiPQ, thus affecting the pervasive transcription. Transcription of rho is autoregulated through expression of both genes of the operon (12). Conversely, it was a Rho-dependent attenuation in the leader region of the transcript.
    [Show full text]
  • Transcription Termination by Nuclear RNA Polymerases
    Downloaded from genesdev.cshlp.org on September 23, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Transcription termination by nuclear RNA polymerases Patricia Richard and James L. Manley1 Department of Biological Sciences, Columbia University, New York, New York 10027, USA Gene transcription in the cell nucleus is a complex and few base pairs to several kilobases downstream from the highly regulated process. Transcription in eukaryotes 39-end of the mature RNA (Proudfoot 1989). RNAPII requires three distinct RNA polymerases, each of which transcription termination is coupled to 39-end processing employs its own mechanisms for initiation, elongation, of the pre-mRNA (Birse et al. 1998; Hirose and Manley and termination. Termination mechanisms vary consid- 2000; Yonaha and Proudfoot 2000; Proudfoot 2004; erably, ranging from relatively simple to exceptionally Buratowski 2005), and an intact polyadenylation signal complex. In this review, we describe the present state of has long been known to be necessary for transcription knowledge on how each of the three RNA polymerases termination of protein-coding genes in human and yeast terminates and how mechanisms are conserved, or vary, cells (Whitelaw and Proudfoot 1986; Logan et al. 1987; from yeast to human. Connelly and Manley 1988). RNAPIII and RNAPI termination appear simpler than Transcription in eukaryotes is performed by three RNA RNAPII termination. RNAPIII terminates transcription polymerases, which are functionally and structurally at T-rich sequences located a short distance from the related (Cramer et al. 2008). RNA polymerase II (RNAPII) mature RNA 39-end and seems to involve at most a is responsible for transcription of protein-coding genes limited number of auxiliary factors (Cozzarelli et al.
    [Show full text]
  • Different Phosphoisoforms of RNA Polymerase II Engage the Rtt103 Termination Factor in a Structurally Analogous Manner
    Different phosphoisoforms of RNA polymerase II PNAS PLUS engage the Rtt103 termination factor in a structurally analogous manner Corey M. Nemeca, Fan Yangb, Joshua M. Gilmorec, Corinna Hintermaird, Yi-Hsuan Hoe,1, Sandra C. Tsenga, Martin Heidemannd, Ying Zhangc, Laurence Florensc, Audrey P. Gasche,f, Dirk Eickd, Michael P. Washburnc,g, Gabriele Varanib, and Aseem Z. Ansaria,f,2 aDepartment of Biochemistry, University of Wisconsin–Madison, Madison, WI 53706; bDepartment of Chemistry, University of Washington, Seattle, WA 98195; cStowers Institute for Medical Research, Kansas City, MO 64110; dDepartment of Molecular Epigenetics, Helmholtz Center Munich, Center for Integrated Protein Science, 81377 Munich, Germany; eLaboratory of Genetics, University of Wisconsin–Madison, Madison, WI 53706; fGenome Center of Wisconsin, University of Wisconsin–Madison, Madison, WI 53706; and gDepartment of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66150 Edited by Alan G. Hinnebusch, National Institutes of Health, Bethesda, MD, and approved April 10, 2017 (received for review January 3, 2017) The carboxyl-terminal domain (CTD) of the largest subunit of RNA Previous studies suggest that pThr4 has roles in transcriptional polymerase II (Pol II) orchestrates dynamic recruitment of specific elongation, 3ʹ-end processing, and termination (12–14,18).Our cellular machines during different stages of transcription. Signature data, as well as other recent studies, suggest that CTD bearing phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of pThr4 is bound by Rtt103 (16, 18), a well-known component of the the CTD engage specific “readers.” Whereas phospho-Ser5 and phos- Rat1 exosome that is thought to play a role in transcription ter- pho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only mination of protein-coding genes.
    [Show full text]
  • Unravelling the Means to an End: RNA Polymerase II Transcription Termination
    REVIEWS Unravelling the means to an end: RNA polymerase II transcription termination Jason N. Kuehner, Erika L. Pearson and Claire Moore Abstract | The pervasiveness of RNA synthesis in eukaryotes is largely the result of RNA polymerase II (Pol II)‑mediated transcription, and termination of its activity is necessary to partition the genome and maintain the proper expression of neighbouring genes. Despite its ever-increasing biological significance, transcription termination remains one of the least understood processes in gene expression. However, recent mechanistic studies have revealed a striking convergence among several overlapping models of termination, including the poly(A)- and Sen1‑dependent pathways, as well as new insights into the specificity of Pol II termination among its diverse gene targets. Broader knowledge of the role of Pol II carboxy‑terminal domain phosphorylation in promoting alternative mechanisms of termination has also been gained. Cryptic unstable transcripts Regulation of gene expression, which is essential for Transcription termination serves many vital func- Non-coding RNAs norma­l cellular development and homeostasis, often tions in the cell. For example, it prevents Pol II from (~200–600 nucleotides long) occurs at the level of transcription. In eukaryotes, nuclear interfering with downstream DNA elements, such as discovered in yeast that are RNA polymerase II (Pol II) is responsible for synthe- promoters, and promotes polymerase recycling4–6. typically transcribed from intergenic regions of the sizing all protein-coding RNAs and most non-coding Contrary to being limited to a constitutive process at genome (such as promoters) RNAs, including small nuclear RNAs (snRNAs), small the end of a gene, transcription termination is dynamic and are rapidly degraded nucleolar RNAs (snoRNAs), microRNAs (mi­RNAs), and can occur upstream, downstream and within open by the exosome.
    [Show full text]
  • Research Progress in the Molecular Functions of Plant Mterf Proteins
    cells Review Research Progress in the Molecular Functions of Plant mTERF Proteins Pedro Robles and Víctor Quesada * Instituto de Bioingeniería, Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-(96)-6658812; Fax: +34-96-665-85-11 Abstract: Present-day chloroplast and mitochondrial genomes contain only a few dozen genes in- volved in ATP synthesis, photosynthesis, and gene expression. The proteins encoded by these genes are only a small fraction of the many hundreds of proteins that act in chloroplasts and mitochondria. Hence, the vast majority, including components of organellar gene expression (OGE) machineries, are encoded by nuclear genes, translated into the cytosol and imported to these organelles. Consequently, the expression of nuclear and organellar genomes has to be very precisely coordinated. Furthermore, OGE regulation is crucial to chloroplast and mitochondria biogenesis, and hence, to plant growth and development. Notwithstanding, the molecular mechanisms governing OGE are still poorly under- stood. Recent results have revealed the increasing importance of nuclear-encoded modular proteins capable of binding nucleic acids and regulating OGE. Mitochondrial transcription termination factor (mTERF) proteins are a good example of this category of OGE regulators. Plant mTERFs are located in chloroplasts and/or mitochondria, and have been characterized mainly from the isolation and analyses of Arabidopsis and maize mutants. These studies have revealed their fundamental roles in different plant development aspects and responses to abiotic stress. Fourteen mTERFs have been hitherto characterized in land plants, albeit to a different extent. These numbers are limited if we consider that 31 and 35 mTERFs have been, respectively, identified in maize and Arabidopsis.
    [Show full text]
  • The Functions of the RNA Polymerase II CTD in Transcription and RNA Processing
    The functions of the RNA polymerase II CTD in transcription and RNA processing Jing-Ping Hsin Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 © 2013 Jing-Ping Hsin All rights reserved Abstract The functions of the RNA polymerase II CTD in transcription and RNA processing Jing-Ping Hsin RNA polymerase II (RNAP II), transcribing messenger RNAs (mRNAs), small nuclear RNAs (snRNAs), and non-coding RNAs (ncRNAs), is composed of 12 subunits. Rpb1, the largest subunit with catalytic polymerase activity, possesses a unique c-terminal domain (CTD) that consists of tandem heptad repeats with the consensus sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser (Y1S2P3T4S5P6S7). Somewhat reflecting the complexity of the organism, the number of repeats varies, from 26 in yeast to 52 in vertebrates. The CTD, intensively phosphorylated during transcription, serves a means to coordinate transcription and RNA processing- capping, splicing, and 3’ end formation. For example, Ser 5, phosphorylated in the start of transcription, promotes the recruitment of capping enzyme, and Ser 2 phosphorylation facilitates RNA 3’ end formation and transcription termination by acting as a landing pad for Pcf11. Detailed introduction is described in Chapter 1. Because of the importance of the CTD in these events, I created an Rpb1 conditional knock-out DT40 cell line (DT40-Rpb1) to further study the CTD with an initial focus on Thr 4, the function of which was unclear. Using DT40-Rpb1 system, we found that Thr 4 was phosphorylated in yeast, fly, chicken, and human cells, and cyclin-dependent kinase (CDK9) was likely the kinase to carry out this phosphorylation.
    [Show full text]