DOCSLIB.ORG

A Family of Activator Genes Regulates Expression of Rhixobium Meliloti

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
A Family of Activator Genes Regulates Expression of Rhixobium Meliloti Copyright 0 1989 by the Genetics Society of America A Family of Activator Genes Regulates Expressionof Rhixobium meliloti Nodulation Genes John T. Mulligan and Sharon R. Long Department of Biological Sciences, Stanford University, Stanford, Calfornia94305-5020 Manuscript received August 29, 1988 Accepted for publication January 27, 1989 ABSTRACT Nodulation (nod) gene expression in Rhizobium meliloti requires plant inducers and the activating protein product of the nodD gene. We have examined three genes in R. meliloti which have nodD activity and sequence homology. These three nodD genes are designated nodD1, nodD2 and nodD3, and have distinctive properties. The nodDl gene product activates expression of the nodABC operon, as measured by a nodC-lacZ fusion or by transcript analysis, in the presence of crude seed or plant wash or the inducer, luteolin. The nodD3 gene product can cause a high basal (uninduced) level of nodC-lacZ expression and nodABC transcripts which is relatively unaffected by inducers. The effect of nodD3 is dependent on the presence of another gene, syrM hmbiotic yegulator). By primer extension analysis we determined that the transcription start site is the same for nodDl plus luteolin or nodD3- syrM mediated expression of nodA and nodH mRNAs. syrM also enhances the expression of another symbiotically important trait, production of extracellular polysaccharide. This regulatory effect of syrM requires locus syrA, which is linked to nodD3 and syrM. The syrM-syrA mediated increase in polysaccharide production requires at least some of the previously identified exo genes and may be a parallel regulatory event to the syrM-nodD3 control of nod promoters. ACTERIA in the genus Rhizobium invade specific tion is under the control of an additional set of sym- B host plants and stimulate the development of biotic genes, the exo genes, are also requiredfor nitrogen-fixing root nodules. This complex and highly normal nodule development(FINAN et al. 1985; CHAK- specific process occurs between a given species of RAVORTY et al. 1982; CHENet al. 1985; BORTHAKUR Rhizobium and a set of plants which defines its host et al. 1986; LEIGH, SIGNERand WALKER1985; DYLAN range. Rhizobium meliloti establishes a productive sym- et al. 1986. Most exo genemutations of R. meliloti biosis with alfalfa (Medicago sativa)and related plants. (deficient in acidic exopolysaccharides) block the nor- The early events in the development of nodules in- mal bacterial invasion of the plant root. When alfalfa clude bacterial stimulation of celldivisions in the plant is inoculated with exo- strains, nodules develop but root cortical layer, giving rise to a new meristem, and few or no infection threads are formed, few if any bacterial invasion of epidermal root hairs and under- bacteria enter the nodules, and no nitrogen fixation lying cells (BAUER198 1; NEWCOMB198 1). occurs (FINANet al. 1985). At least three sets of R. meliloti genes are involved A family of regulatory genes, which are referred to in mediating the early stages of alfalfa nodule forma- as nodD, has been identified in all Rhizobium species tion. One set, nodABC, is present inall Rhizobium so far examined (MULLIGANand LONG 1985; EGEL- species so far examined, and mutationsin these genes HOFF and LONG1985; ROSSENet al. 1985; GOTTFERT lead to a Nod- phenotype: the bacteria neither stim- et al. 1986; SPAINKet al. 1987a, b; SHEARMANet al. ulate cortical cell divisions, nor deform orinvade root 1986). Although some Rhizobium species carry only a hairs (ROSTASet al. 1986; DEBELLEet al. 1986; DUD- single copy of nodD (DOWNIEet al. 1984; SCHOFIELD LEY, JACOBSand LONG 1987; JACOBS,EGELHOFF and and WATSON1985), many species carry two or three LONG 1985). The members of a second set of nodu- similar but not identical copies of nodD (RODRIGUEZ- lation genes (in R. meliloti, nodFE, nodH, nodG, nodPQ QUINONESet al. 1987; APPELBAUMet al. 1988). and others) are notnecessarily present in or function- Expression of nodABC, nodFE and nodH is induced in ally conserved among all Rhizobium species; these af- the presence of an exudate of legume seeds or roots; fect the rate and frequency of nodule formation and this activation requires a nodD gene product. R. meli- influence host range (HORVATHet al. 1986; DJORD- loti has three copies of nodD, designated nodDl, nodD2 JEVIC, SCHOFIELDand ROLFE 1985; DEBELLEet al. and nodD3 (PUTNOKYand KONDOROSI1986; GOTT- 1986; RODRIGUEZ-QUINONESet al. 1987; SWANSONet FERT et al. 1986; HONMAand AUSUBEL1987). al. 1987; SCHWEDOCKand LONG 1989). The flavone, luteolin, isolated from alfalfa seed The Rhizobium exopolysaccharides, whose produc- wash, is the most active inducer of R. meliloti nod Genetics 122: 7-18 (May, 1989) 8 J. T. Mulligan and S. R. Long genes when nodDl is overexpressed (PETERS,FROST of pRmJT5 carrying an insertion of Tn5 which was pro- and LONG 1986).A wide variety of flavones and duced as described in SWANSONet al. (1987) and screened as described in RESULTS. The construction of pRmM 113 flavanones are present in legume exudates, some of involved two steps; first, pRmS507 was digested with ClaI which are active in inducing nod gene expression,and and the fragment bearingthe Tn5 insertion was cloned into others in blocking induction (DJORDEVICet al. 1987; the ClaI site of pBR322. The resulting plasmid was digested REDMONDet al. 1986; FIRMINet al. 1986; ZAAT et al. with Sal1 and XhoI, and the fragment carrying syrM and a 1987; PETERSand LONG 1988). Several lines of evi- segment of Tn5 which encodes neomycin resistance was ligated into pLAFR3 digested with BamHI, by the method dence indirectly suggest that the nodD gene products of partial fill-in of each cohesive end (ZABROVSKYand AL- interact with the inducing molecules. First, the substi- LIKMETS 1986), toform pRmM 1 13.pRmS5 1 1was digested tution of the naturally occurring nodD gene of one with XhoI and religated at low concentrationto form Rhizobium species for the nodD gene of another species pRmM 136, which lacksthe sequences between the Tn5 and in otherwise isogenic strains can determine the range the left-most XhoI site of pRmJT5. pRmM137 (FISHERet al. 1988) was digested with XbaI, partially filled-in and ligated of compounds which will induce expression of the nod to a partially filled-in Hind111 fragment which carries the genes (SPAINKet al. 1987a).Second, a mutation in Tn5 neomycin resistance gene to formpRmM 14 1. pRmJT5 nodD can broaden the spectrum of plant or synthetic was digested with XbaI, partially filled-in and ligated to a compounds which will induce nod gene expression partially filled in Hind111 fragment carrying the uidA gene (BURN,ROSSEN and JOHNSTON 1987). Third, the spe- and spectinomycin resistance to form pRmM142. A 2-kb BglII fragment from pRmS5 1 1was purified and cloned into cies source of nodD in a transgenic construct in some the BamHI site of pUCll9to create pRmM151 and cases influences or determines the host range of the pRmM152. A 2.1-kb ClaI fragmentfrom pRmS511 was Rhizobium strain (SPAINKet al. 1987b; HORVATHet al. cloned into theAccI site of pUCll9 toestablish pRmM 147. 1987; GYORGYPAL, IYERand KONDOROSI 1988). How- Strain construction: Marker exchange was carried out as ever, there are as yet no direct biochemical demon- described by JACOBS,EGELHOFF and LONG (1985) except that pR751 was used to exclude the IncP plasmids. Trans- strations of NodD-inducer interaction.The nodD gene ductions were carried out using phage N3 as described in product, NodD, appears to beDNA-binding a protein MARTINand LONG(1 984).JM 139 was produced by marker by the criterion thatNodD-containing extracts (HONG exchange of the spectinomycin resistance insertion in et al. 1987) and substantially purified NodD (FISHER pRmM139 into R. meliloti 1021. JM80 and JM86 were et al. 1988) affect the electrophoretic mobility of nod produced by marker exchange of the Tn5 insertions from pRmS303 and pRmS701, respectively, into JM6l. JM81 gene promoters. Competition studies show that this and JM83 were produced by marker exchange of the Tn5 binding occursat least partially at thenod box (FISHER insertions from pRmS9B7 and pRmS7O 1, respectively, into et al. 1988), a conserved sequence found upstream of JM57.JM85 was produced by markerexchange of the each inducible nod operon (ROSTASet al. 1986; SCHO- neomycin resistance insertion of pRmM141 intoJM57. FIELD and WATSON 1986; SPAINK et al. 1987b). Tran- JM88, JM204 and JM207 were produced by transduction of the Tn5 insertions from JT303, JT801 and JT709, scription of the inducible nod genes initiates 26-28 bp respectively, into JM57. JM216 was produced by transduc- downstream from the nod box (FISHERet al. 1988). tion of the Tn5 insertion from JT80 1 into JM6 1. JM90 was In this study, we examined the regulatory proper- produced by cotransduction of the nodD3 Tn5 insertion and ties of the threeR. meliloti nodD genes. We found that the nodD1-lac2 fusion of JM80 into JM139. JM96 was pro- while each of the nodD genes is capable of affecting duced by cotransduction of the syrM Tn5 insertion and the nodD1-lacZ fusion of JM86 into JM139. JM201 was pro- the expression of a node-lac2 fusion, the three genes duced by cotransduction of the nodD3 Tn5 insertion and are not equivalent. the nodC-lacZ fusion of JM88 into JM139. The described nodD? requires a second gene, syrM (symbiotic reg- insertions in all of the strains were confirmed by SOUTHERN ulator), for its activating function, but is not depend- blot analysis. ent on flavones to activate expression of nodC-lacZ. Assays: @-galactosidaseassays were carried out as de- scribed by MILLER(1 972) with the modifications previously The transcription start sites for both nodA and nodH described (MULLIGANand LONG1985). Four replicate sam- are unchanged, whether their expression is mediated ples were assayed for each condition and each strain. The by nodDl and luteolin, or by nodD? and syrM. syrM @-galactosidaseunits reported in the text and tables are the can affect both the expression of the nod genes and average of the results from the four assays.
Load more

Recommended publications
  • The Activator Protein-1 Transcription Factor in Respiratory Epithelium Carcinogenesis
    Subject Review The Activator Protein-1 Transcription Factor in Respiratory Epithelium Carcinogenesis Michalis V. Karamouzis,1 Panagiotis A. Konstantinopoulos,1,2 and Athanasios G. Papavassiliou1 1Department of Biological Chemistry, Medical School, University of Athens, Athens, Greece and 2Division of Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts Abstract Much of the current anticancer research effort is focused on Respiratory epithelium cancers are the leading cause cell-surface receptors and their cognate upstream molecules of cancer-related death worldwide. The multistep natural because they provide the easiest route for drugs to affect history of carcinogenesis can be considered as a cellular behavior, whereas agents acting at the level of gradual accumulation of genetic and epigenetic transcription need to invade the nucleus. However, the aberrations, resulting in the deregulation of cellular therapeutic effect of surface receptor manipulation might be homeostasis. Growing evidence suggests that cross- considered less than specific because their actions are talk between membrane and nuclear receptor signaling modulated by complex interacting downstream signal trans- pathways along with the activator protein-1 (AP-1) duction pathways. A pivotal transcription factor during cascade and its cofactor network represent a pivotal respiratory epithelium carcinogenesis is activator protein-1 molecular circuitry participating directly or indirectly in (AP-1). AP-1–regulated genes include important modulators of respiratory epithelium carcinogenesis. The crucial role invasion and metastasis, proliferation, differentiation, and of AP-1 transcription factor renders it an appealing survival as well as genes associated with hypoxia and target of future nuclear-directed anticancer therapeutic angiogenesis (7). Nuclear-directed therapeutic strategies might and chemoprevention approaches.
    [Show full text]
  • Repressor to Activator Switch by Mutations in the First Zn Finger of The
    Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 7086-7090, August 1991 Biochemistry Repressor to activator switch by mutations in the first Zn finger of the glucocorticoid receptor: Is direct DNA binding necessary? (interleukin 1 indudbility/dexamethasone modulation/DNA-binding domain mutants/interleukin 6 and c-fos promoters) ANURADHA RAY, K. STEVEN LAFORGE, AND PRAVINKUMAR B. SEHGAL* The Rockefeller University, New York, NY 10021 Communicated by Igor Tamm, May 20, 1991 (receivedfor review March 15, 1991) ABSTRACT Transfection ofHeLa cells with cDNA vectors previous experiments in HeLa cells transfected with cDNA expressing the wild-type human glucocorticoid receptor (GR) vectors constitutively expressing the wild type (wt) but not enabled dexamethasone to strongly repress cytokine- and sec- the inactive carboxyl-terminal truncation mutants of GR, we ond messenger-induced expression of cotransfected chimeric observed that dexamethasone (Dex) strongly repressed the reporter genes containing transcription regulatory DNA ele- induction by interleukin 1 (IL-1), tumor necrosis factor, ments from the human interleukin 6 (IL-6) promoter. Deletion phorbol ester, or forskolin of IL-6-chloramphenicol acetyl- of the DNA-binding domain or of the second Zn finger or a transferase (CAT) constructs containing IL-6 DNA from point mutation in the Zn catenation site in the second finger -225 to +13. Dex also repressed induction of IL-6- blocked the ability of GR to mediate repression of the IL-6 thymidine kinase (TK)-CAT chimeric constructs containing promoter.
    [Show full text]
  • MINIREVIEW Catabolite Gene Activator Protein Activation of Lac Transcription WILLIAM S
    JOURNAL OF BACTERIOLOGY, Feb. 1992, p. 655-658 Vol. 174, No. 3 0021-9193/92/030655-04$02.00/0 Copyright © 1992, American Society for Microbiology MINIREVIEW Catabolite Gene Activator Protein Activation of lac Transcription WILLIAM S. REZNIKOFF Department of Biochemistry, College ofAgricultural and Life Sciences, University of Wisconsin-Madison, 420 Henry Mall, Madison, Wisconsin 53706 CAP ACTIVATION OF lac TRANSCRIPTION upon binding, and this could lead to contact of upstream DNA with RNA polymerase (21, 25). What is the mechanism by which genes are positively Finally, CAP acts as a repressor in some systems (18, 26). regulated? How can several unlinked genes encoding related Since the lac promoter region (and other regulatory regions functions be regulated by a common signal? These are two of such as gal) contains several promoterlike elements which the questions which can be addressed by studying the overlap the promoter (Fig. 1), it was thought that CAP could catabolite gene activator protein (CAP). CAP responds to activate transcription by limiting the access of nonproduc- differences in the availability and nature of carbon sources, tive competitive promoterlike elements to RNA polymerase via variations in the intracellular concentration of cyclic (16). AMP (cAMP). CAP, when complexed with cAMP, is a sequence-specific DNA-binding protein which activates sev- WHY DIRECT CAP-RNA POLYMERASE CONTACTS eral gene systems and represses others. It has been most ARE PROBABLY IMPORTANT FOR lac ACTIVATION extensively studied for Escherichia coli, although closely related proteins exist in other gram-negative bacteria. Several lines of evidence indicate that direct CAP-RNA CAP is an important paradigm for understanding the polymerase contacts play an important role in lac activation.
    [Show full text]
  • Molecular Biology and Applied Genetics
    MOLECULAR BIOLOGY AND APPLIED GENETICS FOR Medical Laboratory Technology Students Upgraded Lecture Note Series Mohammed Awole Adem Jimma University MOLECULAR BIOLOGY AND APPLIED GENETICS For Medical Laboratory Technician Students Lecture Note Series Mohammed Awole Adem Upgraded - 2006 In collaboration with The Carter Center (EPHTI) and The Federal Democratic Republic of Ethiopia Ministry of Education and Ministry of Health Jimma University PREFACE The problem faced today in the learning and teaching of Applied Genetics and Molecular Biology for laboratory technologists in universities, colleges andhealth institutions primarily from the unavailability of textbooks that focus on the needs of Ethiopian students. This lecture note has been prepared with the primary aim of alleviating the problems encountered in the teaching of Medical Applied Genetics and Molecular Biology course and in minimizing discrepancies prevailing among the different teaching and training health institutions. It can also be used in teaching any introductory course on medical Applied Genetics and Molecular Biology and as a reference material. This lecture note is specifically designed for medical laboratory technologists, and includes only those areas of molecular cell biology and Applied Genetics relevant to degree-level understanding of modern laboratory technology. Since genetics is prerequisite course to molecular biology, the lecture note starts with Genetics i followed by Molecular Biology. It provides students with molecular background to enable them to understand and critically analyze recent advances in laboratory sciences. Finally, it contains a glossary, which summarizes important terminologies used in the text. Each chapter begins by specific learning objectives and at the end of each chapter review questions are also included.
    [Show full text]
  • Solutions for Practice Problems for Molecular Biology, Session 5
    Solutions to Practice Problems for Molecular Biology, Session 5: Gene Regulation and the Lac Operon Question 1 a) How does lactose (allolactose) promote transcription of LacZ? 1) Lactose binds to the polymerase and increases efficiency. 2) Lactose binds to a repressor protein, and alters its conformation to prevent it from binding to the DNA and interfering with the binding of RNA polymerase. 3) Lactose binds to an activator protein, which can then help the RNA polymerase bind to the promoter and begin transcription. 4) Lactose prevents premature termination of transcription by directly binding to and bending the DNA. Solution: 2) Lactose binds to a repressor protein, and alters its conformation to prevent it from binding to the DNA and interfering with the binding of RNA polymerase. b) What molecule is used to signal low glucose levels to the Lac operon regulatory system? 1) Cyclic AMP 2) Calcium 3) Lactose 4) Pyruvate Solution: 1) Cyclic AMP. Question 2 You design a summer class where you recreate experiments studying the lac operon in E. coli (see schematic below). In your experiments, the activity of the enzyme b-galactosidase (β -gal) is measured by including X-gal and IPTG in the growth media. X-gal is a lactose analog that turns blue when metabolisize by b-gal, but it does not induce the lac operon. IPTG is an inducer of the lac operon but is not metabolized by b-gal. I O lacZ Plac Binding site for CAP Pi Gene encoding β-gal Promoter for activator protein Repressor (I) a) Which of the following would you expect to bind to β-galactosidase? Circle all that apply.
    [Show full text]
  • Tuning an Activator-Repressor Clock Employing Retroactivity
    Tuning an Activator-Repressor Clock Employing Retroactivity Alexander Rosenberg∗, Shridhar Jayanthiy and Domitilla Del Vecchioz ∗Electrical Engineering Dept., University of Washington, Seattle WA 98195 yElectrical Engineering and Computer Science Dept., University of Michigan, Ann Arbor MI 48109 zDept. of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge MA 02139 Abstract— Activator-repressor systems have been shown to (a) Activator-Repressor Motif be capable of oscillations and are therefore an important clock motif in the field of Synthetic and Systems Biology. In this paper, we propose a method to regulate oscillatory behavior in such systems by the addition of DNA binding sites for the proteins involved in the clock network. We show that the retroactivity (b) Activator-Repressor with Activator Binding Sites effect caused by this addition can effectively change the relative timescales among the protein dynamics and impact the behavior of the clock. We also employ root locus analysis to obtain a graphical interpretation of the results. I. INTRODUCTION (c) Activator-Repressor with Repressor Binding Sites The design and analysis of oscillating modules is im- portant in the fields of Systems and Synthetic Biology as it enables the understanding of oscillator mechanisms that regulate essential natural processes, such as the cell cycle [1] or circadian clocks [2]. Oscillators are also a useful module in Synthetic Biology as they allow for synchroniza- Fig. 1. Diagram (a) illustrates the activator-repressor motif. Diagram (b) tion of different modules leading to more complex design and (c) illustrate the systems after the addition of DNA binding sites with affinity to the activator and the repressor respectively.
    [Show full text]
  • How Do Eukaryotie Activator Proteins Stimulate the Rate of Transcription by RNA Polymerase II?
    Volunle 307, number 1, 81-86 FEBS 11244 July 1992 0 1992 Federation of European Biochemical Societies 00145793/92/~5.00 Minireview How do eukaryotie activator proteins stimulate the rate of transcription by RNA polymerase II? Jonathan Ham, Gertrud Steger and Moshe Yaniv Uniti des Wrus Otrcog~nes, DGpurtemetrtdes Biotecbtologies, Institut Pusteur, _35 rue du Dr. Ram, 75724 Park Cedex 15, France Received 18 May t992 A large number of activator proteins have now been identified in higher and lower eukaryotes, which bind to the regulatory rcpjons of protein. encoding genes and increase the rate at which tbcy are transcribed by RNA polymerasc II. The mechanism by which activators function is being intensively studied and some of the targets of transcriptional activation domains have now been identified. These studies have also revealed novel classes of regulatory factors, which were not anticipated by extrapolating from the principles obtaira!! with prokaryotic promoters. Activator: RNA polymerasc 11; Mechauism of activation; General transcription faclor; Coactivalor 1. INTRODUCTION 2. THE TRANSCRlPTION INITIATION COM- PLEX A major goal in the field of eukaryotic gene regula- tion is to understand how the activator proteins that The region of a promoter in which the RNA polym- bind to the upstream promoter elements and enhancers erase 11 transcription initiation complex is assembled of RNA polymerase II-dependent genes stimulate the Aridwhere transcription initiates is known as the core rate at which these genes are transcribed. RNA polym- or minimal promoter (Fig. 1). Usually this contains a erase II is unable to recognize promoters on its own and TATA box sequence [3], which specifies the direction of is assisted by a number of accessory proteins, referred transcription and the site of initiaiion, and which will to as the general transcription factors.
    [Show full text]
  • Regulated Expression of the GAL4 Activator Gene in Yeast Provides a Sensitive Genetic Switch for Glucose Repression (Repressors/Weak Promoters/Synergism) DAVID W
    Proc. Natl. Acad. Sci. USA Vol. 88, pp. 8597-8601, October 1991 Genetics Regulated expression of the GAL4 activator gene in yeast provides a sensitive genetic switch for glucose repression (repressors/weak promoters/synergism) DAVID W. GRIGGS AND MARK JOHNSTON Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110 Communicated by Ronald W. Davis, June 21, 1991 ABSTRACT Glucose (catabolite) repression is mediated is presumably due to unidentified repressors that bind to this by multiple mechanisms that combine to regulate transcription region. of the GAL genes over at least a thousandfold range. We have UAS-mediated repression is characterized by the failure of determined that this is due predominantly to modest glucose GAL4 to bind the UAS in cells growing in the presence of repression (4- to 7-fold) of expression of GAL4, the gene glucose (6, 7). This could be due to glucose-induced modifi- encoding the transcriptional activator of the GAL genes. GALA cations ofGAL4 that affect DNA binding, to glucose-induced regulation is affected by mutations in several genes previously proteolysis of the GAL4 protein, or to glucose repression of implicated in the glucose repression pathway; it is not depen- GALA gene expression. We describe experiments that show dent on GAL4 or GAL80 protein function. GALA promoter that GALA expression is modestly reduced by glucose sequences that mediate glucose repression were found to lie through the action of specific negatively acting elements in downstream of positively acting elements that may be "TATA the GALA promoter. The resulting reduction in intracellular boxes." Two nearly identical sequences (10/12 base pairs) in GAL4 activator levels leads to a greatly amplified effect on this region that may be binding sites for the MIG1 protein were expression of GAL] and accounts for a substantial portion of identified as functional glucose-control elements.
    [Show full text]
  • Mechanisms of Prokaryotic Gene Regulation
    Overview: Conducting the Genetic Orchestra • Prokaryotes and eukaryotes alter gene expression in response to their changing environment • In multicellular eukaryotes, gene expression regulates development and is responsible for differences in cell types • RNA molecules play many roles in regulating gene expression in eukaryotes Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 18-1 1 Concept 18.1: Bacteria often respond to environmental change by regulating transcription • Natural selection has favored bacteria that produce only the products needed by that cell • A cell can regulate the production of enzymes by feedback inhibition or by gene regulation • Gene expression in bacteria is controlled by the operon model Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings Fig. 18-2 Precursor Feedback inhibition trpE gene Enzyme 1 trpD gene Regulation of gene expression Enzyme 2 trpC gene trpB gene Enzyme 3 trpA gene Tryptophan (a) Regulation of enzyme (b) Regulation of enzyme activity production 2 Operons: The Basic Concept • A cluster of functionally related genes can be under coordinated control by a single on-off “switch” • The regulatory “switch” is a segment of DNA called an operator usually positioned within the promoter • An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings • The operon can be switched off by a protein repressor • The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase • The repressor is the product of a separate regulatory gene Copyright © 2008 Pearson Education Inc., publishing as Pearson Benjamin Cummings 3 • The repressor can be in an active or inactive form, depending on the presence of other molecules • A corepressor is a molecule that cooperates with a repressor protein to switch an operon off • For example, E.
    [Show full text]
  • Transcription Activation by Catabolite Activator Protein (CAP)
    ArticleNo.jmbi.1999.3161availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (1999) 293, 199±213 Transcription Activation by Catabolite Activator Protein (CAP) SteveBusby1andRichardH.Ebright2* 1School of Biosciences, The Transcription activation by Escherichia coli catabolite activator protein University of Birmingham (CAP) at each of two classes of simple CAP-dependent promoters is Birmingham B15 2TT, UK understood in structural and mechanistic detail. At class I CAP-depen- dent promoters, CAP activates transcription from a DNA site located 2Howard Hughes Medical upstream of the DNA site for RNA polymerase holoenzyme (RNAP); at Institute, Waksman Institute these promoters, transcription activation involves protein-protein inter- and, Department of Chemistry actions between CAP and the RNAP a subunit C-terminal domain that Rutgers University, New facilitate binding of RNAP to promoter DNA to form the RNAP-promo- Brunswick, NJ 08855, USA ter closed complex. At class II CAP-dependent promoters, CAP activates transcription from a DNA site that overlaps the DNA site for RNAP; at these promoters, transcription activation involves both: (i) protein-protein interactions between CAP and RNAP a subunit C-terminal domain that facilitate binding of RNAP to promoter DNA to form the RNAP-promo- ter closed complex; and (ii) protein-protein interactions between CAP and RNAP a subunit N-terminal domain that facilitates isomerization of the RNAP-promoter closed complex to the RNAP-promoter open com- plex. Straightforward combination of the mechanisms for transcription activation at class I and class II CAP-dependent promoters permits syner- gistic transcription activation by multiple molecules of CAP, or by CAP and other activators. Interference with determinants of CAP or RNAP involved in transcription activation at class I and class II CAP-dependent promoters permits ``anti-activation'' by negative regulators.
    [Show full text]
  • Yeast GCN4 Transcriptional Activator Protein Interacts with RNA
    Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2652-2656, April 1989 Biochemistry Yeast GCN4 transcriptional activator protein interacts with RNA polymerase II in vitro (gene regulation/promoters/affinity chromatography/mRNA initiation/eukaryotic transcription) CHRISTOPHER J. BRANDL AND KEVIN STRUHL Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115 Communicated by Howard Green, February 1, 1989 ABSTRACT Regulated transcription by eukaryotic RNA related to the jun oncoprotein (15-17), an oncogenic version polymerase II (Pol II) requires the functional interaction of of the vertebrate AP-1 transcription factor (18, 19). multiple protein factors, some of which presumably interact It has been proposed that GCN4, like other yeast activator directly with the polymerase. One such factor, the yeast GCN4 proteins, stimulates transcription by directly contacting other activator protein, binds to the upstream promoter elements of components of the transcriptional machinery (5, 13, 20, 21). many amino acid biosynthetic genes and induces their tran- Evidence against the idea that upstream activator proteins scription. Through the use of affinity chromatography involv- function by increasing chromatin accessibility comes from ing GCN4- or Pol II-Sepharose columns, we show that GCN4 the observation that GAL4 cannot stimulate transcription by interacts specifically with Pol II in vitro. Purified Pol II is bacteriophage T7 RNA polymerase in yeast (22). In contrast, retained on the GCN4-Sepharose column under conditions in a poly(dA-dT) sequence, which is hypothesized to cause a which the vast majority of proteins flow through. Moreover, local disruption in chromatin structure, enhances transcrip- Pol II can be selectively isolated from more complex mixtures tion by T7 RNA polymerase (22).
    [Show full text]
  • Silencing of Tonebp/NFAT5 Transcriptional Activator by RNA Interference
    J Am Soc Nephrol 14: 283–288, 2003 Silencing of TonEBP/NFAT5 Transcriptional Activator by RNA Interference KI YOUNG NA, SEUNG KYOON WOO, SANG DO LEE, and H. MOO KWON Division of Nephrology, Johns Hopkins University, Baltimore, Maryland. Abstract. TonEBP is a transcriptional activator that is ex- sion and expression of the sodium/myo-inositol cotransproter pressed throughout development in many tissues and cell (SMIT), aldose reductase (AR) and heat shock protein 70 types. In the kidney medulla, TonEBP appears to be an impor- (HSP70) mRNA were significantly decreased in cells where tant local regulator of differentiation by virtue of stimulating TonEBP expression was silenced. These data provide direct several genes. To study the function of TonEBP, two small evidence that the SMIT, AR, and HSP70 genes are targets of interfering RNA (siRNA) duplexes were developed that re- TonEBP, although the potential role of other proteins, such as duced TonEBP expression effectively via RNA interference. accessory proteins, cannot be excluded. The TonEBP-siRNA is The silencing lasted only 3 d after introduction of the TonEBP- an effective tool that should prove useful in the investigation of siRNA’s. As expected, TonEBP-driven reporter gene expres- loss-of-function relationship in cells. TonEBP (tonicity-responsive enhancer binding protein) is a accumulation of urea (UT-A), protection of cells from the high transcriptional activator of the Rel family that includes NF-␬B concentration of urea (HSP70). In cultured cells, TonEBP is and NFAT. The Rel family proteins share unique structural stimulated by an increase in ambient tonicity in temporal features in the DNA binding domains.
    [Show full text]