DOCSLIB.ORG

Interaction of the Operator of the Tryptophan Operon with Repressor (Repressor-Operator Interactions/Trp Repressor/In Vitro Transcription/Escherichia Coli) JOHN K

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Interaction of the Operator of the Tryptophan Operon with Repressor (Repressor-Operator Interactions/Trp Repressor/In Vitro Transcription/Escherichia Coli) JOHN K Proc. Nat. Acad. Sci. USA Vol. 71, No. 8, pp. 3134-3138, August 1974 Interaction of the Operator of the Tryptophan Operon with Repressor (repressor-operator interactions/trp repressor/in vitro transcription/Escherichia coli) JOHN K. ROSE AND CHARLES YANOFSKY Department of Biological Sciences, Stanford University, Stanford, California 94305 Contributed by Charles Yanofsky, April 23, 1974 ABSTRACT Transcription studies in vitro on repres- where stated otherwise, the ratio of polymerase to DNA was sion ofthe tryptophan operon of Escherichia coli show that maintained at 1:1 (w/w). Each reaction mixture also con- partially purified trp repressor binds specifically to DNA containing the trp operator with a repressor-operator tained 6 M1A of buffer IV (1) with the indicated amounts of dissociation constant of about 0.2 nM in 0.12 M salt at partially purified repressor. Buffer IV added final concentra- 370, a value consistent with the extent of trp operon tions of 2 mM Tris-OAc (pH 8.0), 12 mM KOAc, 2.5 mM regulation in vivo. The half-life of the trp repressor-trp Mg(OAc)2, 0.14 mM dithiothreitol, and 1% glycerol. RNA operator complex is less than 2 min in vitro in 0.12 M salt. synthesis, RNA purification, and DNA template preparation Partially purified trp aporepressor (1) represses transcription were as described (2). The initial steps of aporepressor puri- in vitro of the tryptophan (trp) operon of Escherichia coli in the fication were as described (1), and then the aporepressor was presence of itryptophan or appropriate tryptophan analogues taken through an additional DEAE-cellulose column step (2-4). Neither tryptophanyl-tRNA synthetase (3, 5) nor which gave 10-fold further purification (5). The final prepara- tryptophanyl-tRNA (5-7) appears to participate in repression. tion of aporepressor was estimated to be 550-fold purified over Repression in vitro involves interaction between the trp that in crude extracts. This preparation contains about 1 nM repressor and the trp operator, since an operator constitutive aporepressor at a protein concentration of 5.2 sg/ml (Fig. 2). mutation eliminates repression (2). On the basis of these find- Assuming a molecular weight of 58,000 (1, 5), the active apo- ings it may be tentatively concluded that the protein product repressor would constitute about 1.0% of the protein in the of trpR, the aporepressor, when complexed with the corepres- preparation. sor, L-tryptophan, acts at the trp operator to present transcrip- RESULTS of tion the operon. The template DNA used in most of the transcription experi- Attempts to assay trp repressor-trp operator interaction from a specialized assays (8) have been ments in vitro described here was isolated by conventional DNA-protein binding transducing phage, 080 trpO+ AtrpEDl02 trpCBl, which unsuccessful. Therefore, we have used the in vitro transcrip- of trpA, and this interaction. carries the trp operator, trpC, trpB, and a portion tion system to study has a particular deletion of trpE and D. This deletion results MATERIALS AND METHODS in relatively high rates of trpCB mRNA synthesis in vivo and Bacteriophage Isolation. The transducing phages 480 trpO+ in vitro (10, 2). trpCB mRNA is detected by hybridization of AtrpED102 trpCBl and XtrpCBl have been described (2). synthesized labeled mRNA to the separated strands of the carries and the operator proximal DNA of XtrpCB1. In agreement with previous results (2), we Phage 080 trpO+ trpE7 trpE from the portion of trpD and was isolated after induction of an E. coli find that approximately 8% of the RNA transcribed the deletion tonB trpAD32. This dele- 4)80 trpO+ AtrpEDl02 trpCBl template by purified RNA poly- 480h- lysogen carrying correct or c strand of XtrpCB1 DNA, tion removes the operator distal genes trpA, trpB, and trpC, merase hybridizes to the carries the trp and less than 1% hybridizes to the incorrect or i strand of the and about 80% of trpD. Phage 480 trpO+ trpE7 to X as shown formation anal- latter DNA. A blank value of 0.3-0.4% hybridization operator and promoter, by enzyme In ex- similar to those described by DNA has been subtracted from these percentages. all yses in infection experiments in excess Franklin (9). The gene order in the operon is trp operator (0)- periments described below, tryptophan was present at a concentration of 0.3 mM, 30-fold above that required for E-D-C-B-A. half-maximal repression (2). Reaction Conditions In Vitro and Repressor Preparation. In vitro reactions (2) were for 15 min at 37°. The final volume Determination of the dissociation constant of each reaction mixture was 30 /l, containing 0.3 mM L- Fig. 1 shows the percent repression of correct strand trpCB tryptophan, 12 mM Tris- HC1 (pH 7.9), 1.3 mM MgCI2, 97 mRNA synthesis at various repressor concentrations. Each mM KCl, 0.13 mM EDTA, 0.1 mnM dithiothreitol, 0.09 mM curve presents results obtained at a different template DNA ATP, CTP, and GTP, 0.017 mM [3H]UTP (10 Ci/mmole), concentration with the ratio of polymerase to DNA held con- 10 /Ag/ml (0.33 nM) to 150 ,g/ml (5 nM) of DNA and 10 /ug/ stant. Note that as the DNA concentration is increased (at ml to 153 ,ug/ml of RNA polymerase holoenzyme (EC 2.7.7.6; repressor preparation levels of less than 8 ,ug/ml), there is a nucleosidetriphosphate: RNA nucleotidyltransferase). Except decrease in percent repression of trp operon transcription. Note also that at low DNA concentrations a change in DNA Abbreviations: trp, tryptophan gene or operon; c strand and i concentration has only a relatively small effect on repression, strand, correct and incorrect strand, respectively, of XtrpCB1 while at higher DNA concentrations (>1 nM) the effect is DNA. large. For example, a repressor concentration that is just 3134 Downloaded by guest on September 28, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) trp Operon Regulation 3135 sufficient to give 50% repression with 0.33 nM DNA shows only a 1.3-fold reduction in percent repression (to 38%) when the DNA concentration is increased 3-fold to 1 nM. A much larger (3.8-fold) reduction in repression (to 10%) is seen when the DNA concentration is increased another 5-fold to 5 nM. These results suggest that at the low DNA concentrations .2a tested there are significant levels of free repressor and free 0co,ID operator. Thus, if we assume that percent repression indicates 4e the percent of operators present that are bound to repressor, 10% repression of 5 nM DNA would be equivalent to a repres- sor concentration of at least 0.5 nM. This same repressor con- centration, however, gives only 50% repression of 0.33 nM DNA, although the repressor concentration must be higher than the DNA concentration. Therefore, in this case, we con- clude that more than half of the repressor is not bound to trp Partially Purified Repressor (mg/ml) operators. Similar reasoning leads to the conclusion that the FIG. 1. Repression of trp operon transcription as a function of of the molecules are bound to DNA and repressor concentrations. Reaction conditions in vitro majority trp repressor present and partially purified repressor were as described in Materials and trp operators at the higher DNA concentrations used in these Methods. The indicated concentrations of partially purified experiments. The above observations suggest an equilibrium repressor and 0.3 mM L-tryptophan were preincubated with between trp repressor, trp operator, and the trp repressor-trp template DNA for 2 min at 00 in the standard reaction mixture. operator complex, with the value for the dissociation constant RNA polymerase was added and incubation was continued for of the complex near the concentrations of repressor and opera- anadditional 2min at 00. Reactions were initiated by addition tor. of nucleoside triphosphates and were run for 15 min at 37°. We shall show below that these data are consistent with a Template (080 trpO+ AtrpED102 trpCBl DNA) concentrations simple equilibrium situation described as follows:* were: A, 0.33 nM; A, 0.5 nM; 0, 1 nM; *, 2 nM; 0, 2.5 nM; X, 5 nM. The RNA polymerase:DNA ratio was constant at each R + 0 2 RO [1] DNA concentration. Total 3H cpm in RNA ranged from 18,651 (0.33 nM DNA) to 285,033 (5 nM DNA). At each DNA con- The dissociation constant at equilibrium for this reaction is centration, trp CB c strand mRNA (measured by hybridization to XtrpCBl c strand DNA) was 6-8% of the total RNA synthe- K -[R -RO] [0-RO] [2] sized. The repressor fraction used has no activity without trypto- [RO] phan and does not affect either phage transcription or i strand where R = total repressor, 0 = total operator, [R - RO] = trp transcription. Longer preincubations of repressor with DNA free repressor, [O - RO I = free operator, and [RO] = re- (370) at the lowest repressor and DNA concentrations do not pressor-operator ] enhance repression, indicating that repressor-operator association complex. Equation [2 can be expressed as reaches equilibrium within 2 min at 00. K = 1 X [ 0]- (1 -X) [RI [3] where [01 = the operator concentration that saturates any given fraction X of repressor [X = (RO/R)]. For example, assumed to indicate the percent of total operators bound to when the operator concentration gives one-half saturation of repressor molecules. The total repressor concentration is deter- R, i.e., when RO = 1/2 R and X = '/2, equation [3] becomes mined from the plateau values in experiments in which the operator concentration is increased until the concentration of K = [01/2 - 1/2[R] [4] repressed operators reaches a plateau.
Load more

Recommended publications
  • Transcriptional Units
    Proc. Natl. Acad. Sci. USA Vol. 76, No. 7, pp. 3194-3197, July 1979 Biochemistry Cyclic AMP as a modulator of polarity in polycistronic transcriptional units (positive regulation/rho factor/lactose and galactose operons/catabolite repression) AGNES ULLMANNt, EVELYNE JOSEPHt, AND ANTOINE DANCHINt tUnite de Biochimie Cellulaire, Institut Pasteur, 75724 Paris Cedex 15, France; and tInstitut de Biologie Physico-Chimique, 75005 Paris, France Communicated by Frangois Jacob, April 16, 1979 ABSTRACT The degree of natural polarity in the lactose scribed by Watekam et al. (7, 8) in sonicated bacterial extracts. and galactose operons of Escherichia coli is affected by aden- One unit is the amount of enzyme that converts 1 nmol of osine 3',5'-cyclic monophosphate (cAMP). This effect, mediated substrate per min at 280C (except for UDPGal epimerase, for by the cAMP receptor protein, is exerted at sites distinct from the promoter. Experiments performed with a mutant bearing which the assay temperature was 220C). a thermosensitive rho factor activity indicate that cAMP relieves Reagents and Enzymes. They were obtained from the fol- polarity by interfering with transcription termination. Con- lowing companies: trimethoprim from Calbiochem; all radio- flicting results in the literature concerning the role of cAMP active products from Amersham; isopropyl-f3-D-thiogalactoside receptor protein and cAMP in galactose operon expression can (IPTG), D-fucose, cAMP, UDPglucose dehydrogenase, and all be reconciled by the finding that cAMP stimulates the expres- substrates from Sigma; and all other chemicals from Merck. sion of operator distal genes without significantly affecting the proximal genes. Therefore, it appears necessary to reevaluate the classification o(the galactose operon as exhibiting cAMP- RESULTS mediated catabolite repression at the level of transcription Natural Polarity in Lactose Operon.
    [Show full text]
  • Sudips Revised Thesis
    Investigation Of The Behavior Of The Gal4 Inhibitor Gal80 Of The GAL Genetic Switch In The Yeast Saccharomyces Cerevisiae Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Sudip Goswami, M.S. Graduate Program in Molecular Genetics The Ohio State University 2014 Dissertation Committee Dr. James Hopper, Advisor Dr. Stephen Osmani Dr. Hay-Oak Park Dr. Jian-Qiu Wu ii Copyright by Sudip Goswami 2014 iii ABSTRACT The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to upstream activation sequences or UASGAL sites on GAL gene promoters as a dimer both in the absence and presence of galactose. In the absence of galactose, a Gal80 dimer binds to and masKs the Gal4 activation domain, inhibiting its activity. In the presence of galactose, Gal3 interacts with Gal80 and relieves Gal80’s inhibition of Gal4 activity allowing rapid induction of expression of GAL genes. In the first part of this work (Chapter 2) in-vitro chemical crosslinking coupled with SDS PAGE and native PAGE analysis were employed to show that the presence of Gal3 that can interact with Gal80 impairs Gal80 self association. In addition, live cell spinning disK confocal imaging showed that dissipation of newly discovered Gal80-2mYFP/2GFP clusters in galactose is dependent on Gal3’s ability to interact with Gal80. In the second part (Chapter 3), extensive analysis of Gal80 clusters was carried out which showed that these clusters associate strongly with the GAL1-10-7 locus and this association is dependent on the presence of the UASGAL sites at this locus.
    [Show full text]
  • NADP, Corepressor for the Bacillus Catabolite Control Protein Ccpa
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 9590–9595, August 1998 Microbiology NADP, corepressor for the Bacillus catabolite control protein CcpA JEONG-HO KIM,MARTIN I. VOSKUIL, AND GLENN H. CHAMBLISS* Department of Bacteriology, University of Wisconsin-Madison, E. B. Fred Hall, Madison, WI 53706 Communicated by T. Kent Kirk, University of Wisconsin, Madison, WI, June 10, 1998 (received for review February 12, 1998) ABSTRACT Expression of the a-amylase gene (amyE)of (18), and the presence of a conserved effector-binding domain Bacillus subtilis is subject to CcpA (catabolite control protein in CcpA (3) together strongly suggest the necessity of an A)-mediated catabolite repression, a global regulatory mech- effector(s) such as a corepressor to activate CcpA. To date, anism in Bacillus and other Gram-positive bacteria. To deter- HPr, a phosphocarrier protein in the phosphoenolpyruvate- mine effectors of CcpA, we tested the ability of glycolytic :sugar phosphotransferase system, and two glycolytic metab- metabolites, nucleotides, and cofactors to affect CcpA binding olites, fructose-1,6-diphosphate (FDP) and glucose-6- to the amyE operator, amyO. Those that stimulated the phosphate, have been proposed as effectors of CcpA (19–22). DNA-binding affinity of CcpA were tested for their effect on HPr is phosphorylated in two different fashions: ATP- transcription. HPr-P (Ser-46), proposed as an effector of dependent phosphorylation at Ser-46 and phosphoenolpyru- CcpA, also was tested. In DNase I footprint assays, the affinity vate-dependent phosphorylation at His-15 (23). In the ptsH1 of CcpA for amyO was stimulated 2-fold by fructose-1,6- mutant strain, the serine residue at position 46 of HPr is diphosphate (FDP), 1.5-fold by oxidized or reduced forms of replaced with an alanine, which eliminates phosphorylation of NADP, and 10-fold by HPr-P (Ser-46).
    [Show full text]
  • Studies with the Escherichia Coli Galactose Operon Regulatory Region Carrying a Point Mutation That Simultaneously Inactivates T
    CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Volume 219, number 1, 189-196 FEB 04899 July 1987 Studies with the Escherichia coli galactose operon regulatory region carrying a point mutation that simultaneously inactivates the two overlapping promoters Interactions with RNA polymerase and the cyclic AMP receptor protein Sreenivasan Ponnambalarn, Annick Spassky* and Stephen Busby Department of Biochemistry, University of Birmingham, PO Box 363, Birmingham B15 2TT, England and *D~partement de Biologie Molbculaire, lnstitut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France Received 22 April 1987 We report in vitro studies of the interactions between purified E. coli RNA polymerase and DNA from the regulatory region of the E. coli galactose operon which carries a point mutation that simultaneously stops transcription initiation at the two normal start points, $1 and $2. In the presence of this point muta- tion, transcription initiates at a third start point 14/15 bp downstream of $1, showing that inactivation of the two normally active promoters, P1 and P2, unmasks a third weaker promoter, P3. Transcription initia- tion in the gal operon is normally regulated by the cyclic AMP receptor protein, CRP, that binds to the gal regulatory region and switches transcription from P2 to PI. With the point mutation, CRP binding swit- ches transcription from P3 to P1, although the formation of transcriptionally competent complexes at P1 is very slow. The results are discussed with respect to the mechanism of transcription activation by the CRP factor and the similarities between the regulatory regions of the galactose and lactose operons.
    [Show full text]
  • Chapter 21 Operons: Fine Control of Bacterial Transcription
    Chapter 21 Operons: Fine Control of Bacterial Transcription The E. coli genome contains over 3000 genes. Some of these are active all the time because their products are in constant demand. But some of them are turned off most of the time because their products are rarely needed. For example, the enzymes required for the metabolism of the sugar arabinose would be useful only when arabinose is present and when the organism’s favorite energy source, glucose, is absent. Such conditions are not common, so the genes encoding these enzymes are usually turned off. Why doesn’t the cell just leave all its genes on all the time, so the right enzymes are always there to take care of any eventuality? The reason is that gene expression is an expensive process. It takes a lot of energy to produce RNA and protein. In fact, if all of an E. coli cell’s genes were turned on all the time, production of RNAs and proteins would drain the cell of so much energy that it could not compete with more effi cient organisms. Thus, control of gene expression is essential to life. Gene regulation is the device to insure that proteins are synthesized in exactly the amounts they are needed and only when they are needed. Transcriptional regulation: gene expression is controlled by regulatory proteins Negative regulation: - A repressor protein inhibits transcription of a specific gene. - In this case, inducer (antagonist of the repressor) is needed to allow transcription. Positive regulation: - Activator works to increase the frequency of transcription of an gene (operon) Transcriptional regulation: gene expression is controlled by regulatory proteins Operons Operons and the resulting transcriptional regulation of gene expression permit prokaryotes to rapidly adapt to changes in the environment: new carbon sources, lack of an amino acid, etc.
    [Show full text]
  • The Regulation of Transcription Initiation in Bacteria
    Annual Reviews www.annualreviews.org/aronline Ann.Rev. Genet. 1985. 19:35547 Copyright© 1985 by AnnualReviews Inc. All rights reserved THE REGULATION OF TRANSCRIPTIONINITIATION IN BACTERIA William S. Reznikoff I, Deborah A. Siegele 2, Deborah W. Cowingz, and2 Carol A. Gross Departmentsof Biochemistry~ andBacteriology 2, Collegeof Agriculturaland Life Sciences, Universityof Wisconsin,Madison, Wisconsin 53706 CONTENTS INTRODUCTION..................................................................................... 355 PROMOTERSTRUCTURE ......................................................................... 356 THEREGULATION OF E~TM TRANSCRIPTIONINITIATION ............................ 360 NegativeRegulation of TranscriptionInitiation ............................................. 361 Positive Regulationof TranscriptionInitiation .............................................. 363 DNATopology Regulation of TranscriptionInitiation ...................................... 365 DNAModification and the Regulationof GeneExpression ............................... 366 MODIFICATION OF HOLOENZYMESTRUCTURE AND THE REGULATION OF GENEEXPRESSION ................................................................... 366 E. coil ~r7° .......................................................................................... 367 ~2 by University of Wisconsin - Madison on 03/29/07. For personal use only. The HeatShock Response and ~r ............................................................. 368 The E. coli ntrA (glnF) Protein: AnotherSigma Factor ..................................
    [Show full text]
  • Molecular Mechanisms of Transcription Initiation at Gal Promoters and Their Multi-Level Regulation by Galr, CRP and DNA Loop
    Biomolecules 2015, 5, 2782-2807; doi:10.3390/biom5042782 OPEN ACCESS biomolecules ISSN 2218-273X www.mdpi.com/journal/biomolecules/ Review Molecular Mechanisms of Transcription Initiation at gal Promoters and their Multi-Level Regulation by GalR, CRP and DNA Loop Dale E.A. Lewis * and Sankar Adhya Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4255, USA; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-301-496-9129; Fax: +1-301-496-2212. Academic Editors: Sivaramesh Wigneshweraraj and Deborah M. Hinton Received: 11 May 2015 / Accepted: 25 September 2015 / Published: 16 October 2015 Abstract: Studying the regulation of transcription of the gal operon that encodes the amphibolic pathway of D-galactose metabolism in Escherichia coli discerned a plethora of principles that operate in prokaryotic gene regulatory processes. In this chapter, we have reviewed some of the more recent findings in gal that continues to reveal unexpected but important mechanistic details. Since the operon is transcribed from two overlapping promoters, P1 and P2, regulated by common regulatory factors, each genetic or biochemical experiment allowed simultaneous discernment of two promoters. Recent studies range from genetic, biochemical through biophysical experiments providing explanations at physiological, mechanistic and single molecule levels. The salient observations highlighted here are: the axiom of determining transcription start points, discovery of a new promoter element different from the known ones that influences promoter strength, occurrence of an intrinsic DNA sequence element that overrides the transcription elongation pause created by a DNA-bound protein roadblock, first observation of a DNA loop and determination its trajectory, and piggybacking proteins and delivering to their DNA target.
    [Show full text]
  • Isolation of the Gal Repressor (E
    Proc. Nat. Acad. Sci. USA Vol. 68, No. 8, pp. 1891-1895, August 1971 Isolation of the gal Repressor (E. coli/lac repressor/fucose/galactose/affinity chromatography) J. S. PARKS*, M. GOTTESMAN*, K. SHIMADAt, R. A. WEISBERGt, R. L. PERLMAN$, AND I. PASTAN* * Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health;t Laboratory of Biomedical Sciences, National Institute of Child Health and Human Development, National Institutes of Health; and $ Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Maryland 20014 Communicated by E. R. Stadtman, June 11, 1971 ABSTRACT The repressor of the galactose operon of I867Sam7) (6); Xh8Odlacp8 from PP70, F-,strRA(lac-proA),- Escherichia coli has been partially purified and identified Su- (Xh8OdlacpscJ857St68,Xh8Ocl857St68) (9); and X from as a protein. Induction of a lysogen in which X was linked N1383, Su-,galK-, Cultures were grown in to the bacterial gaiR and lysine genes resulted in a large (XcI857Sam7). increase in the production of the gal repressor. Single-step IL broth (10 g Bacto-tryptone-5 g yeast extract-5 g NaCl purification by affinity chromatography, using the ligand per liter) and induced by heating log-phase cultures at 40'C p-aminophenyl-B3-D-thiogalactoside linked to beaded agar- for 3-4 hr. Isolation and purification of bacteriophage and ex- ose, provided a convenient method of separating the gal traction of bacteriophage DNA have been described (10). To repressor from other DNA-binding proteins. Binding of gal repressor to Xpgal [32PJDNA was studied by assay of binding label phage DNA, 5 mCi Of 32p was added to 100 ml of cul- to a nitrocellulose filter.
    [Show full text]
  • Spot 42 RNA Mediates Discoordinate Expression of the E. Coli Galactose Operon
    Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Spot 42 RNA mediates discoordinate expression of the E. coli galactose operon Thorleif Møller, Thomas Franch, Christina Udesen, Kenn Gerdes, and Poul Valentin-Hansen1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark The physiological role of Escherichia coli Spot 42 RNA has remained obscure, even though the 109-nucleotide RNA was discovered almost three decades ago. Structural features of Spot 42 RNA and previous work suggested to us that the RNA might be a regulator of discoordinate gene expression of the galactose operon, a control that is only understood at the phenomenological level. The effects of controlled expression of Spot 42 RNA or deleting the gene (spf) encoding the RNA supported this hypothesis. Down-regulation of galK expression, the third gene in the gal operon, was only observed in the presence of Spot 42 RNA and required growth conditions that caused derepression of the spf gene. Subsequent biochemical studies showed that Spot 42 RNA specifically bound at the galK Shine-Dalgarno region of the galETKM mRNA, thereby blocking ribosome binding. We conclude that Spot 42 RNA is an antisense RNA that acts to differentially regulate genes that are expressed from the same transcription unit. Our results reveal an interesting mechanism by which the expression of a promoter distal gene in an operon can be modulated and underline the importance of antisense control in bacterial gene regulation. [Key Words: Antisense RNA; galactose operon; riboregulation; small RNAs; Spot 42RN A; translational regulation] Received March 22, 2002; revised version accepted May 13, 2002.
    [Show full text]
  • Region Immediately Adjacent to the L-Arabinose Operator in Escherichia Coli B/R' MARIANNE ELEUTERIO, BARBARA GRIFFIN, and DAVID E
    JOURNAL OF BACTERIOLOGY, Aug. 1972. p. 383-391 Vol. 111, No. 2 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Characterization of Strong Polar Mutations in a Region Immediately Adjacent to the L-Arabinose Operator in Escherichia coli B/r' MARIANNE ELEUTERIO, BARBARA GRIFFIN, AND DAVID E. SHEPPARD Department of Biological Sciences, University of Delaware, Newark, Delaware 19711 Received for publication 12 April 1972 Seven L-arabinose-negative mutations are described that map in three genet- ically distinct regions immediately adjacent to the araO (operator) region of the L-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dom- inant, trans-recessive polarity effect upon the expression of L-arabinose isom- erase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initi- ator region of the L-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens di- ethyl sulfate and ICR-191. Three of these mutants are suppressible by two in- dependently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara+ revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppres- sors, and their partially polar revertants are discussed in terms of the mecha- nism of initiation of expression of the L-arabinose operon.
    [Show full text]
  • Local and Global Regulation of Transcription Initiation in Bacteria Browning, Douglas F; Busby, Stephen J W
    University of Birmingham Local and global regulation of transcription initiation in bacteria Browning, Douglas F; Busby, Stephen J W DOI: 10.1038/nrmicro.2016.103 License: None: All rights reserved Document Version Peer reviewed version Citation for published version (Harvard): Browning, DF & Busby, SJW 2016, 'Local and global regulation of transcription initiation in bacteria', Nature Reviews Microbiology. https://doi.org/10.1038/nrmicro.2016.103 Link to publication on Research at Birmingham portal Publisher Rights Statement: Checked 11/08/2016 General rights Unless a licence is specified above, all rights (including copyright and moral rights) in this document are retained by the authors and/or the copyright holders. The express permission of the copyright holder must be obtained for any use of this material other than for purposes permitted by law. •Users may freely distribute the URL that is used to identify this publication. •Users may download and/or print one copy of the publication from the University of Birmingham research portal for the purpose of private study or non-commercial research. •User may use extracts from the document in line with the concept of ‘fair dealing’ under the Copyright, Designs and Patents Act 1988 (?) •Users may not further distribute the material nor use it for the purposes of commercial gain. Where a licence is displayed above, please note the terms and conditions of the licence govern your use of this document. When citing, please reference the published version. Take down policy While the University of Birmingham exercises care and attention in making items available there are rare occasions when an item has been uploaded in error or has been deemed to be commercially or otherwise sensitive.
    [Show full text]
  • Escherichia Coli LUCIA B
    JOURNAL oF BAcTERIOLoGY, June 1973, p. 1040-1044 Vol. 114, No. 3 Copyright 0 1973 American Society for Microbiology Printed in U.SA. Role of Cyclic Adenosine 3', 5'-Monophosphate in the In Vivo Expression of the Galactose Operon of Escherichia coli LUCIA B. ROTHMAN-DENES, JOANNE E. HESSE, AND WOLFGANG EPSTEIN Departments of Biophysics and Biochemistry, The University of Chicago, Chicago, Illinois 60637 Received for publication 22 December 1972 Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3', 5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3', 5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3', 5'-monophosphate or of the binding protein for this nucleotide. Genetic (7, 19, 20) and biochemical (2, 4, 6, 7) except for the presence of the araC+ and araCc67 evidence has accumulated in recent years, mutations, respectively. L104 is a strain constitutive showing that the catabolite repression of in- for the fl-methylgalactosidase transport system (13). ducible enzymes is mediated by cyclic adeno- Chemicals. [1-_4C]galactose was obtained from Calbiochem, and [U-_4C]glycerol was obtained from sine 3', 5'-monophosphate (c-AMP) and a pro- International Chemical and Nuclear Corp. tein factor (CRP) that specifically binds c-AMP. Bacterial growth and preparation of extracts.
    [Show full text]