DOCSLIB.ORG

RNA Polymerase Mutations That Impair Conversion to a Termination-Resistant Complex by Q Antiterminator Proteins

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
RNA Polymerase Mutations That Impair Conversion to a Termination-Resistant Complex by Q Antiterminator Proteins Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press RNA polymerase mutations that impair conversion to a termination-resistant complex by Q antiterminator proteins Thomas J. Santangelo,1 Rachel Anne Mooney,2 Robert Landick,2 and Jeffrey W. Roberts1,3 1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York14853, USA; 2Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA Bacteriophage ␭ Q-protein stably binds and modifies RNA polymerase (RNAP) to a termination-resistant form. We describe amino acid substitutions in RNAP that disrupt Q-mediated antitermination in vivo and in vitro. The positions of these substitutions in the modeled RNAP/DNA/RNA ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for Q in RNAP, but instead act by impairing interactions among core RNAP subunits and nucleic acids that are essential for Q modification. A specific conjecture is that Q modification stabilizes interactions of RNAP with the DNA/RNA hybrid and optimizes alignment of the nucleic acids in the catalytic site. Such changes would inhibit the activity of the RNA hairpin of an intrinsic terminator to disrupt the 5؅-terminal bases of the hybrid and remove the RNA 3؅ terminus from the active site. [Keywords: RNA polymerase; antitermination; termination; transcription; Q-protein; bacteriophage ␭] Received February 7, 2003; revised version accepted March 24, 2003. Both specific and general elongation factors promote ␾82, are stably incorporated into RNAP during a pro- transcription through natural barriers in chromosomes. moter-proximal ␴70-dependent pause (Yarnell and Rob- These include the discrete termination sites of prokary- erts 1992; Ring et al. 1996; Roberts et al. 1998). Q-modi- otes, and less well-defined transcription barriers of eu- fied complexes have fundamentally altered elongation karyotes: promoter-proximal pause sites, 3Ј-processing- properties, displaying increased elongation rate, dimin- linked terminators, and nucleosomes. General transcrip- ished pausing, and resistance to all downstream intrinsic tion elongation factors include NusA and NusG of and ␳-dependent termination sites; these properties can bacteria and the highly conserved Spt4/Spt5/Spt6 pro- be expressed in a defined minimal biochemical system teins of eukaryotic Pol II; regulon-specific factors include containing RNAP, Q-protein, and the elongation factor the Q-protein and N-protein of the Escherichia coli bac- NusA. Although the correlation of antipausing and an- teriophage ␭ and the TAT protein of HIV (Roberts 1988; titermination originally suggested a kinetic basis of an- Greenblatt et al. 1993; Laspia et al. 1993; Roberts et al. titermination—namely, that RNAP might elongate 1998). The recent atomic resolution structures of mul- faster than some rate-limiting step of termination—it is tisubunit prokaryotic and eukaryotic RNA polymerases clear that Q induces a more fundamental stabilization of (RNAP), including structures and models of elongation the transcription complex. Thus, static Q-modified elon- complexes, provide an opportunity to describe the activ- gation complexes stopped by a DNA cross-linkat the ity of these factors in molecular detail (Zhang et al. 1999; release site of an intrinsic terminator in vitro are stabi- Cramer et al. 2000; Ebright 2000; Korzheva et al. 2000; lized against the dissociation activity of the terminator Gnatt et al. 2001; Korzheva and Mustaev 2001; Mu- (T.J. Santangelo and J.W. Roberts, unpubl.). Similarly, rakami et al. 2002a,b; Vassylyev et al. 2002). The poly- static Q-modified complexes are stabilized at any elon- merases share a strikingly similar structural core, sug- gation site against attackby an oligonucleotide that gesting that insights into regulatory regions, domains, simulates the activity of an intrinsic terminator (Yarnell and even individual residues may be universal. and Roberts 1999). Finally, Q-modified elongation com- The phage-␭-encoded Q-protein antitermination fac- plexes resist the action of a strong intrinsic terminator tor, and Q-proteins of related phages including ␾21 and when they are transcribing extremely slowly because of limiting NTP concentrations (Roberts et al. 1998). To understand the molecular basis of Q modification, 3Corresponding author. we isolated mutations altering the core subunits of E-MAIL [email protected]; FAX (607) 255-2428. RNAP that interfere with Q function but do not impair Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ ␭ gad.1082103. the basic enzymatic activity of RNAP. We used both Q GENES & DEVELOPMENT 17:1281–1292 © 2003 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/03 $5.00; www.genesdev.org 1281 Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Santangelo et al. and the analogously functioning Q82 from phage ␾82. genized libraries using reporter constructs specific for Although Q␭ and Q82 both modify RNAP at a ␴70-depen- each Q-protein (see Materials and Methods). Briefly, dent promoter-proximal pause, they share no obvious se- these reporters contained the ␭ late promoter upstream quence homology and act only on their own phage pro- of two strong intrinsic terminators, followed by the lac moters with specific associated sequences (Yang et al. operon, making ␤-galactosidase expression Q-dependent. 1989). Presumably, they nonetheless act by the same ba- Mutagenized plasmid libraries of rpoB and rpoC (encod- sic mechanism and may share structural homology. ing the ␤ and ␤Ј subunits, respectively) were introduced To interpret the phenotypes of the mutant RNAPs, we to strains carrying the reporters and the appropriate Q consider the distinctive nature of the interaction be- source, and mutations limiting ␤-galactosidase expres- tween Q and RNAP. Q can only productively bind and sion were isolated. To generate a positive phenotype, the functionally modify RNAP in a paused elongation com- screens require that overproduced plasmid-encoded pro- plex containing the initiating ␴70 subunit; Q makes di- tein incorporate into and produce an active RNAP; the rect contact with a specific site in DNA, with RNAP wild-type genomic copy of rpoB or rpoC was still ex- core subunits, and, for Q␭ at least, with region 4 of ␴70 pressed. RNAP preparations from cells grown under con- (Ko et al. 1998; Nickels et al. 2002). Importantly, after ditions used for the screens show that ∼85%–90% of RNAP clears this pause, Q no longer can bind the un- RNAPs contained the plasmid-encoded His6-tagged sub- modified complex. Productive engagement of Q releases unit (data not shown). RNAP from the pause site and Q becomes a firmly bound We isolated 17 point mutations in rpoB or rpoC that subunit, stable (for Q82 at least) to repeated washing of significantly diminish Q reporter activity: 11 individual immobilized complexes in vitro; ␴70 can (and might usu- mutations, and 3 pairs for which each mutation alone ally) dissociate at this stage. has no phenotype (Fig. 1). Seven mutants were isolated Because of the site-specific and irreversible nature of with each Q reporter, excluding alternative changes at this association, the only step in Q function that is plau- the same site. Of the 17 substitutions, 16 modify the ␤ sibly sensitive to Q concentration is its initial engage- subunit of RNAP; most mutations cluster between or ment with RNAP at the pause site. In fact, most of the within conserved regions D, E, F, or G (Fig. 1B). The defects exhibited by the mutant RNAPs we identified are remaining mutation maps to conserved region G of the rescued partly or completely at increased concentrations ␤Ј subunit. These sites are primarily in domains struc- of Q, implying that these defects are expressed at the turally conserved between Thermus aquaticus RNAP, pause site. Because formation of the paused complex it- Thermus thermophilus RNAP, and Saccharomyces cer- self is not impaired (data not shown), only two sorts of evisiae RNA Pol II ⌬4/⌬7 (Zhang et al. 1999; Cramer et mutational change are likely to be rescued by a higher al. 2000; Ebright 2000; Vassylyev et al. 2002), suggesting concentration of Q. First, amino acid substitutions could that they affect basic elements of the general transcrip- damage a site where Q makes direct contact with the tion process, and could further define the role(s) of in- enzyme. Second, a mutational change in RNAP could dividual domains and residues for all multisubunit disrupt other molecular interactions required for the RNAPs. modification that stably incorporates Q at the pause site. We tested each mutant, regardless of the reporter with The mutational sites that we identified and describe here which it was originally isolated, with both reporters form several discrete clusters in conserved and function- (Figs. 1, 2). Each mutant decreases expression 2–10-fold ally important parts of RNAP that are largely exposed with the reporter for which it was selected, and most within the nucleic acid-binding, catalytic cleft, but reduce expression of both reporters, consistent with a likely are buried and inaccessible to an external protein common mechanism of action of both Q-proteins. How- in the elongation complex. Based on these locations, and ever, some mutants are defective quite specifically for the in vitro properties of the modified enzymes, we argue the reporter with which they were originally selected that
Load more

Recommended publications
  • Galactosidase
    Copyright 0 1988 by the Genetics Society of America Effects of Amino Acid Substitutions atthe Active Site in Escherichia coli @-Galactosidase Claire G. Cupples and Jeffrey H. Miller Molecular Biology Institute and Department of Biology, University of Calqornia, Los Angeles, Calqornia 90024 Manuscript received April 2 1, 1988 Accepted July 23, 1988 ABSTRACT Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme p- galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1 % wild-type activity. His-464 and Met-3 were replaced with 1 1and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-46 1 and Tyr-503 areessential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an fl bacteriophage containing the complete lacz gene on i single ECORIfragment. -GALACTOSIDASE (EC 3.2.1.23) is produced in and J.
    [Show full text]
  • BMB400 Part Four - II = Chpt
    BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymerase B M B 400 Part Four: Gene Regulation Section II = Chapter 17. TRANSCRIPTIONAL REGULATION EXERTED BY EFFECTS ON RNA POLYMERASE [Dr. Tracy Nixon made major contributions to this chapter.] A. The multiple steps in initiation and elongation by RNA polymerase are targets for regulation. 1. RNA Polymerase has to * bind to promoters, * form an open complex, * initiate transcription, * escape from the promoter, * elongate , and * terminate transcription. See Fig. 4.2.1. 2. Summarizing a lot of work, we know that: • strong promoters have high KB, high kf, low kr, and high rates of promoter clearance. • weak promoters have low KB, low kf, high kr, and low rates of promoter clearance. • moderate promoters have one or more "weak" spots. 3. To learn these facts, we need: • genetic data to identify which macromolecules (DNA and proteins) interact in a specific regulation event, and to determine which base pairs and amino acid residues are needed for that regulation event. • biochemical data to describe the binding events and chemical reactions that are affected by the specific regulation event. Ideally, we would determine all forward and reverse rate constants, or equilibrium constants (which are a function of the ratio of rate constants) if rates are inaccessible. Although, in reality, we cannot get either rates or equilibrium constants for many of the steps, some of the steps are amenable to investigation and have proved to be quite informative about the mechanisms of regulation. BMB400 Part Four - II = Chpt. 17. Transcriptional regulation by effects on RNA polymerase Fig.
    [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]
  • Mechanism of Promoter Repression by Lac Repressor–DNA Loops Nicole A
    156–166 Nucleic Acids Research, 2013, Vol. 41, No. 1 Published online 9 November 2012 doi:10.1093/nar/gks1011 Mechanism of promoter repression by Lac repressor–DNA loops Nicole A. Becker1, Justin P. Peters1, Troy A. Lionberger2 and L. James Maher III1,* 1Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, Rochester, MN 55905, USA and 2Howard Hughes Medical Institute and Jason L. Choy Laboratory of Single-Molecule Biophysics, Department of Physics, University of California, Berkeley, CA 94720, USA Received June 29, 2012; Revised October 1, 2012; Accepted October 2, 2012 ABSTRACT presence of allolactose or its analog, isopropyl b-D-1- thiogalactopyranoside (IPTG), relieving repression. In the The Escherichia coli lactose (lac) operon encodes absence of glucose, RNA polymerase binds cooperatively the first genetic switch to be discovered, and lac with catabolite activator protein at the lac promoter (positive remains a paradigm for studying negative and control). In simplest terms, the mechanism of negative positive control of gene expression. Negative control involves Lac repressor binding to occlude access control is believed to involve competition of RNA of RNA polymerase holoenzyme to the lac promoter (4). polymerase and Lac repressor for overlapping Of particular significance to the present work is the binding sites. Contributions to the local Lac repres- fascinating observation that two remote auxiliary oper- sor concentration come from free repressor and re- ators (Oaux) exist in the lac operon (5). It has been pressor delivered to the operator from remote proposed and demonstrated (6–13) that bidentate repres- auxiliary operators by DNA looping.
    [Show full text]
  • Tnlo-Encoded Tet Repressor Can Regulate an Operator-Containing
    Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1394-1397, March 1988 Biochemistry TnlO-encoded tet repressor can regulate an operator-containing plant promoter (cauliflower mosaic virus 35S promoter/electroporation/transient chloramphenicol acetyltransferase assays) CHRISTIANE GATZ* AND PETER H. QUAILt Departments of Botany and Genetics, University of Wisconsin, Madison, WI 53706 Communicated by Folke Skoog, October 26, 1987 (receivedfor review July S, 1987) ABSTRACT The TnlO-encoded tet repressor-operator The TnlO-encoded tet repressor regulates the expression system was used to regulate transcription from the cauliflower of the Tc resistance operon by binding to nearly identical mosaic virus (CaMV) 35S promoter. Expression was moni- operator sequences that overlap with three divergent pro- tored in a transient assay system by using electric field- moters (14, 15). The genes of the tet operon are only mediated gene transfer ("electroporation") into tobacco pro- transcribed in the presence of the inducer Tc, which pre- toplasts. The tet repressor, being expressed in the plant cells vents the repressor from binding to its operator sequences. under the control of eukaryotic transcription signals, blocks The tet repressor was chosen for regulating a plant promoter transcription of a CaMV 35S promoter chloramphenicol ace- for two reasons. (i) With a native molecular mass of 48 kDa, tyltransferase (cat) fusion gene when the two tet operators diffusion into the nucleus seemed likely (16). (ii) The high flank the "TATA" box. In the presence of the inducer equilibrium association constant of the repressor-inducer tetracycline, expression is restored to full activity. Location of complex ensures efficient induction at sublethal Tc concen- the operators 21 base pairs downstream of the transcription trations (17), thus making the system useful as an on/off start site does not significantly affect transcription in the switch for the specific regulation of transferred genes.
    [Show full text]
  • The Lac Operon (BIOT 4006: Genetics and Molecular Biology)
    The lac Operon (BIOT 4006: Genetics and Molecular Biology) Dr. Saurabh Singh Rathore Department of Biotechnology MGCU Structural genes of the lac operon • Two enzymes have important role in lactose metabolism: 1. Permease: acts as a transporter for lactose intake. 2. β-galactosidase: breaks the lactose into glucose and galactose. Lactose metabolism. Hydrolysis of the β-galactoside linkage present in lactose by β-galactosidase results in formation of galactose and glucose. Reference: Introduction to Genetic Analysis, Ninth Edition, Anthony Griffiths et al., Chapter 10 • The adjacent genetic sequences Z and Y encode the β-galactosidase and permease proteins, respectively. • The transacetylase enzyme (not needed in lactose metabolism) is encoded by the gene A. The genes Z, Y and A are called structural genes. • All the three genes give a single mRNA after transcription however the products of Z and Y are important. • The regulation of lac operon is done for synthesis of all the three enzymes simultaneously. That means, either the mRNA is synthesized or not synthesized. • If the different proteins are transcribed by the genes acting as a single transcription unit, the regulation of their expression occurs coordinately. A simple model of lac operon. The expression of the repressor protein by the I gene negatively controls the coordinated expression of the Z, Y, and A genes. Engagement of repressor by inducer results in expression of the structural genes. Reference: Introduction to Genetic Analysis, Ninth Edition, Anthony Griffiths et al., Chapter 10 The lac regulatory components • There are 3 Key regulatory components in the lac operon. One of them is a transcription regulatory protein and the other two are the docking sites for the regulatory protein and the RNA polymerase.
    [Show full text]
  • Saccharomyces Cerevisiae Promoter Engineering Before and During the Synthetic Biology Era
    biology Review Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era Xiaofan Feng and Mario Andrea Marchisio * School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Tianjin 300072, China; [email protected] * Correspondence: [email protected] or [email protected] Simple Summary: Promoters are DNA sequences where the process of transcription starts. They can work constitutively or be controlled by environmental signals of different types. The quantity of proteins and RNA present in yeast genetic circuits highly depends on promoter strength. Hence, they have been deeply studied and modified over, at least, the last forty years, especially since the year 2000 when Synthetic Biology was born. Here, we present how promoter engineering changed over these four decades and discuss its possible future directions due to novel computational methods and technology. Abstract: Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic Citation: Feng, X.; Marchisio, M.A. promoters built via different methods and discuss the latest innovations in the wet-lab engineering Saccharomyces cerevisiae Promoter of novel promoter sequences.
    [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]
  • Allostery and Applications of the Lac Repressor
    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Allostery and Applications of the Lac Repressor Matthew Almond Sochor University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons, and the Biophysics Commons Recommended Citation Sochor, Matthew Almond, "Allostery and Applications of the Lac Repressor" (2014). Publicly Accessible Penn Dissertations. 1448. https://repository.upenn.edu/edissertations/1448 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1448 For more information, please contact [email protected]. Allostery and Applications of the Lac Repressor Abstract The lac repressor has been extensively studied for nearly half a century; this long and complicated experimental history leaves many subtle connections unexplored. This thesis sought to forge those connections from isolated and purified components up ot functioning lac genetic switches in cells and even organisms. We first connected the genetics and structure of the lac repressor to in vivo gene regulation in Escherichia coli. We found that point mutations of amino acids that structurally make specific contacts with DNA can alter repressor-operator DNA affinity and even the conformational equilibrium of the repressor. We then found that point mutations of amino acids that structurally make specific contacts with effector molecules can alter repressor-effector affinity and the conformational equilibrium. Allesults r are well explained by a Monod, Wyman, and Changeux model of allostery. We next connected purified in vitro components with in vivo gene regulation in E. coli. We used an in vitro transcription assay to measure repressor-operator DNA binding affinity, repressor-effector binding affinity, and conformational equilibrium.
    [Show full text]
  • Review Session
    REVIEW SESSION Wednesday, September 15 5:305:30 PMPM SHANTZSHANTZ 242242 EE Gene Regulation Gene Regulation Gene expression can be turned on, turned off, turned up or turned down! For example, as test time approaches, some of you may note that stomach acid production increases dramatically…. due to regulation of the genes that control synthesis of HCl by cells within the gastric pits of the stomach lining. Gene Regulation in Prokaryotes Prokaryotes may turn genes on and off depending on metabolic demands and requirements for respective gene products. NOTE: For prokaryotes, “turning on/off” refers almost exclusively to stimulating or repressing transcription Gene Regulation in Prokaryotes Inducible/RepressibleInducible/Repressible gene products: those produced only when specific chemical substrates are present/absent. ConstitutiveConstitutive gene products: those produced continuously, regardless of chemical substrates present. Gene Regulation in Prokaryotes Regulation may be Negative: gene expression occurs unless it is shut off by a regulator molecule or Positive: gene expression only occurs when a regulator mole turns it on Operons In prokaryotes, genes that code for enzymes all related to a single metabolic process tend to be organized into clusters within the genome, called operonsoperons. An operon is usually controlled by a single regulatory unit. Regulatory Elements ciscis--actingacting elementelement: The regulatory region of the DNA that binds the molecules that influences expression of the genes in the operon. It is almost always upstream (5’) to the genes in the operon. TransTrans--actingacting elementelement: The molecule(s) that interact with the cis-element and influence expression of the genes in the operon. The lac operon The lac operon contains the genes that must be expressed if the bacteria is to use the disaccharide lactose as the primary energy source.
    [Show full text]
  • The Nucleotide Sequence of the Lac Operator
    Proc. Nat. Acad. Sci. USA Vol. 70, No. 12, Part I, pp. 3581-3584, December 1973 The Nucleotide Sequence of the lac Operator (regulation/protein-nucleic acid interaction/DNA-RNA sequencing/oligonucleotide priming) WALTER GILBERT AND ALLAN MAXAM Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138 Communicated by J. D. Watson, Augut 9, 1973 ABSTRACT The lac repressor protects the lac operator bind again to the repressor, and is about 27 base-pairs long. against digestion with deoxyribonuclease. The protected Here we shall describe its sequence. fragment is double-stranded and about 27 base-pairs long. We determined the sequence of RNA transcription METHODS copies of this fragment and present a sequence for 24 base pairs. It is: Sonicated DNA Fragments. Sonicated [82P]DNA fragments 5'--T GG AATT GT GA GC GG AT AAC AATT 3' were made by growing a temperature-inducible lysogen of 3'--AC C TT AACA C TC GC C T ATT GTT AA5' Xcl857plac5S7 at 340 in a glucose-50 mM Tris HCl or TES The sequence has 2-fold symmetry regions; the two longest (pH 7.4) medium in 3 mM phosphate, heating at 420 for 15 are separate4 by one turn ofthe DNA double helix. min at a cell density of 4 X 108/mI, then washing and resus- pending the cells at a density of 8 X 108/ml in the same me- The lactose repressor selects one out of six million nucleotide dium with 0.1 mM phosphate. 100 mCi of neutralized H3s2PO4 sequences in the Escherichia coli genome and binds to it to was added to 10 ml of cells, and the incorporation was con- prevent the expression of the genes for lactose metabolism.
    [Show full text]
  • 1 Biol 3301 Genetics Exam #2A October 26, 2004
    Biol 3301 Genetics Exam #2A October 26, 2004 This exam consists of 40 multiple choice questions worth 2.5 points each, for a total of 100 points. Good luck. Name____________________________________ SS#_____________________________________ 1. Which of the following statements is false: Answer: e a) SWI-SNF activates transcription by moving nucleosomes from the promoter. b) HAT acetylates histones to activate transcription. c) HATs and HDACs modify the acetylation of lysines residues. d) HDAC reverses transcriptional activation due to HATs e) Lowering the amount of histones also lowers gene transcription 2. Methylation of _______________ decreases gene transcription, and is the basis of imprinting. Answer: b a) adenine b) cytosine c) guanine d) thymine e) uracil 3. Which of the following transcription factors enhance transcription without binding to DNA? Answer: a a) Coactivator b) Architectural c) TFIID d) Pol II e) Zinc finger proteins 4. A transcription factor binds to the sequence TAAGCTAAGCCCTTG and inhibits transcription. What functional domains must this protein contain? (Note: no other transcription factor can bind this sequence) a) Dimerization, DNA binding and activation. Answer: b b) Repression and DNA binding. c) Repression, dimerization and DNA binding. d) DNA binding and activation. e) DNA binding. 5. Which cis sequence can function within an intron to regulate transcription? Answer: c a) CCAAT box b) TATA box c) enhancer d) GC-rich box e) Shine-Dalgarno sequence 6. The arabinose operon can only be expressed under the following conditions: Answer: b a) High levels of glucose, low levels of cAMP, low levels of arabinose. b) Low levels of glucose, high levels of cAMP, high levels of arabinose.
    [Show full text]