DOCSLIB.ORG

Identification of Repressor Binding Sites Controlling Expression of Tetracycline Resistance Encoded by Tnjo LEWIS V

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Identification of Repressor Binding Sites Controlling Expression of Tetracycline Resistance Encoded by Tnjo LEWIS V JOURNAL OF BACTERIOLOGY, Dec. 1983, p. 1188-1191 Vol. 156, No. 3 0021-9193/83/121188-04$02.00/0 Copyright © 1983, American Society for Microbiology Identification of Repressor Binding Sites Controlling Expression of Tetracycline Resistance Encoded by TnJO LEWIS V. WRAY, JR.,t AND WILLIAM S. REZNIKOFF* Department ofBiochemistry, College ofAgricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 Received 23 May 1983/Accepted 7 September 1983 The regulatory region controlling the expression of tetracycline resistance and repressor genes contains two nearly identical regions of dyad symmetry. Dele- tions of this control region were isolated by digestion with S1 nuclease. The ability of these deletions to bind the tet repressor was determined by an in vivo repressor titration assay. The results indicate that repressor specifically binds both regions of dyad symmetry. The tetracycline resistance determinant asso- MATERIALS AND METHODS ciated with the plasmid R100 is part of a trans- Materials. Growth media were prepared as de- posable genetic element called transposon TnJO scribed by Miller (24). Concentrations of antibiotics (16). Resistance does not result from the inacti- (Sigma Chemical Co.) used were 60 jig of kanamycin vation of the antibiotic (17). Instead, the resist- per ml and 100 ,g of ampicillin per ml. ance mechanism appears to involve the active Restriction enzymes were purchased from Bethesda efflux of tetracycline from the cell (1, 21). In Research Laboratories or New England Biolabs and addition, decreased ribosomal sensitivity to tet- were used as described by the manufacturer. S1 nucle- racycline may contribute to resistance ase and T4 polynucleotide kinase were obtained from (17, 28). P-L Biochemicals, Inc. T4 DNA ligase was a gift from The expression of tetracycline resistance is R. Simoni. regulated; preincubation with a subinhibitory Bacterial strains. All bacterial strains were de- concentration of tetracycline increases the level rivatives of Escherichia coli K-12. Strain JD600 is of resistance (13). TnJO directs the synthesis of leu hsdM hsdR tonA supE lacY thi. Strain MZ12 is two tetracycline-inducible proteins with appar- Alac(ipozy)X74 trp thi. ent molecular weights of 36,000 and 25,000 (26- Plasmids and phage. Plasmids pBR322, pACYC177, 28). The largest is referred to as the TET protein and pRT86 have been described previously (5, 6, 26). and is known to be negatively regulated by a Plasmid pRZ4045 contains a lac promoter fragment repressor which is inactive in the cloned between the EcoRI and Sall sites of pBR322 presence of (R. Johnson, M. Peterson, and W. Reznikoff, unpub- tetracycline (27). The TET protein is membrane- lished data). Plasmid DNA was isolated by the method associated and is essential for tetracycline resist- of Humphreys et al. (12). ance (15, 27). The 25-kilodalton protein is the The bacteriophage XRS205 is a promoter cloning repressor and has been shown to negatively vector that contains a lacZ gene derived from a trp-lac regulate its own synthesis (2, 26). The location fusion strain, W205, that deletes the lacPO region but of the structural genes for both of these proteins leaves the lacZ gene intact. This phage contains a has been determined (7, 15, 26) (Fig. 1). single EcoRI site and a single Sall site located between These two genes are transcribed divergently the phage att site and the promoterless lacZ gene. A from a common control region has promoter-bearing DNA fragment inserted into these which been sites controls the expression of lacZ. The construction shown to bind purified RNA polymerase (15) of this vector will be described elsewhere (K. P. and tet repressor (9). The DNA sequence of this Bertrand, K. Postle, L. V. Wray, Jr., and W. S. regulatory region has been determined and has Reznikoff, manuscript in preparation). revealed the presence of two nearly identical Plasmid and deletion constructions. A plasmid clone regions of dyad symmetry (4, 10). The experi- of the repressor gene was constructed by digesting ments described in this report demonstrate that pRT86 and pACYC177 with HincIl. After heat inacti- these symmetrical sequences are the repressor vation of the restriction enzyme, the DNA was treated binding sites. with T4 DNA ligase and used to transform JD600 cells by the method of Mandel and Higa (18). The cells were inoculated into 4 ml of LB medium and incubated at t Present address: Institut fur Organische Chemie und 37°C with shaking for 90 min. Transformants were Biochemie, Technische Hochschule Darmstadt, D-6100 selected by plating on tryptone-yeast extract plates Darmstadt, Federal Republic of Germany. containing kanamycin. Since the HinclI site of 1188 VOL. 156, 1983 tet REPRESSOR BINDING SITES 1189 TET protein DNA was self-ligated with T4 DNA ligase and used for .| 1 i695695~~~PO,iN I transformation as described above. Transformants were selected by plating on tryptone-yeast extract repre plates containing ampicillin. tetracycline DNA sequencing. Deletions generated from the SalI site were end labeled at the XbaI restriction site by inactive repressor treatment with [,y-32P]ATP (Amersham Corp.) and FIG. 1. Physical and genetic map of transposon polynucleotide kinase. After digestion with BgII, the TnlO. The thick lines represent the inverted repeat labeled fragments were isolated and the sequence sequence of ISIO, and unlabeled vertical lines are determined by the method of Maxam and Gilbert (20). HincII cleavage sites. The structural gene for the The deletions generated from the XbaI restriction site repressor is located entirely within the 695-base-pair were cloned as EcoRI-SalI fragments into the single- HinclI fragment. P, Promoter; 0, operator. stranded M13 cloning vector Mp8 (23) and were se- quenced by the method of Sanger et al. (25). 3-Galactosidase assays. Cells were grown in M9 pACYC177 lies within the gene encoding f3-lactamase, minimal medium supplemented with 0.2% glucose, 80 the transformants were screened for sensitivity to ,ug of tryptophan per ml, 4 ,ug of thiamine per ml, and ampicillin. The presence of the HincIl 695 fragment antibiotics where appropriate. Cultures were assayed was confirmed by electrophoretic analysis of HincIl by the method of Miller (24). digests of the resulting plasmids. The orientation of the insertions was determined by comparing the restric- RESULTS tion patterns from a double digestion with BamHI and XbaI. pRT241 contains the TnlO HincIl 695 fragment Construction of recombinant plasmids and inserted so that the tet repressor and ,B-lactamase phage. In an earlier study (26), the tet repressor genes are in opposite orientations. gene was mapped to the TnWO HincII 695 frag- Deletions of the tet regulatory region were con- ment. To construct a plasmid encoding the re- structed with pRT301 digested with either XbaI or pressor, this fragment was inserted into the SalI. Linearized plasmid DNA (20 ,ug) was treated HincII site of pACYC177. The resulting plas- with 100 units of Si nuclease at 28°C in a buffer mid, pRT241, is resistant to kanamycin and is consisting of 25 mM NaCl-3 mM ZnSO4-25 mM as sodium acetate (pH 5.0). Portions were removed after compatible with ColEl replicons such 5, 10, 15, 20, 25, and 30 min of incubation, and pBR322. This plasmid does not contain the reactions were terminated by extraction with phenol. operator sites controlling the expression of the After extraction with ether, the samples were dialyzed repressor, and thus, repressor synthesis is un- against 10 mM Tris (pH 7.9) with Millipore VMWP regulated. filters as described by Marusyk and Sergeant (19). The The plasmid pRT301 has the 160-base-pair Kan TnIO tot repressor XRT301 Sal - EcoRI A. I loc z te tt A500 ci857 S FIG. 2. Structure of plasmids and phage. The TnlO portions in the different plasmids are shown as well as the location of the kanamycini (Kan) and ampicillin (Amp) resistance genes. 1190 WRAY AND REZNIKOFF J. BACTERIOL. TABLE 1. Repressor titration slight increase in the level of ,B-galactosidase. Plasmidsa P-Galactosidase activity' When the pBR322 tet promoter region was re- moved, as in pRZ4045, this increase was not None ..................... 5,080 observed. pACYC177 ...... 5,160 Isolation and sequence determination of dele. pRT241 ..................... 206 tions of the tet control region. To determine the pRT241 and pBR322 .......... 293 DNA sequence required for repressor binding pRT241 and pRZ4045 ......... 209 pRT241 and pRT301 .......... 3,730 activity, a series of plasmids containing dele- pRT241 and pRT315 .......... 3,870 tions of the tet control region in pRT301 were pRT241 and pRT317.......... 1,700 prepared. The endpoints of the various deletions pRT241 and pRT320 .......... 202 are shown in Fig. 3. pRT241 and pRT323 .......... 1,630 The deletions 315, 317, 320, and 323 were pRT241 and pRT331 .......... 3,590 generated by Si nuclease digestion from the pRT241 and pRT332.......... 2,700 XbaI site, whereas deletions 331, 332, 333, 336, pRT241 and pRT333 .......... 1,210 and 339 were generated from the SalI site. and .......... 1,230 pRT241 pRT336 Effect of deletions on repressor binding. Plas- pRT241 and pkT339 .......... 208 mids containing the deletions of the tet control a All assays were performed in strain MZ12 which is region were analyzed for their ability to bind lysogenic for XRT301. repressor by the repressor titration assay (Table b 3-Galactosidase activity is in units described by 1). Miller (24). Values given are the average from three independent cultures. All assays were repeated at least Plasmids pRT317 and pRT323 had an interme- twice. diate level of repressor titration. These plasmids contain deletions of all or part of 01, a region of dyad symmetry, without extending into 02, the TaqI fragment containing the tet control region second region of dyad symmetry. Plasmid cloned between the EcoRI and Sall sites of pRT320, which contains a deletion extending pBR322 (Fig. 2). This EcoRI-SalI fragment was into 02, does not have the ability to bind repres- cloned into the promoter cloning vector XRS205 sor.
Load more

Recommended publications
  • A Region of Phage Lambda Required for Antitermination of Transcription
    Cell, Vol. 31, 61-70, November 1982, Copyright 0 1982 by MIT Analysis of nutR; A Region of Phage Lambda Required for Antitermination of Transcription Eric R. Olson, Eric L. Flamm* and 1971; Friedman and Baron, 1974; Keppel et al., David I. Friedman 1974; Friedman et al., 1976, 1981; Greenblatt et al., Department of Microbiology and Immunology 1980). The sites include putative sites of pN action, The University of Michigan Medical School called nut (Salstrom and Szybalski, 1978; Rosenberg Ann Arbor, Michigan 48109 et al., 1978), as well as termination signals (Roberts, 1969; Rosenberg et al., 1978; Rosenberg and Court, 1979). Summary Consideration of the nature of the various factors involved in pN action formed the basis for the following The N gene product of coliphage lambda acts with model of pN action (discussed by Friedman and host factors (Nus) through sites (not) to render Gottesman, 1982). Transcription initiating at the early subsequent downstream transcription resistant to promoters PR and P, extends through the n&R and a variety of termination signals. These sites, not!? nutl sites, respectively (Figure 1). At these sites RNA and nutL, are downstream, respectively, from the polymerase is modified, rendering continuing tran- early promoters PR and PL. Thus a complicated set scription resistant to termination signals. The nature of molecular interactions are likely to occur at the of the promoter appears to play no role in pN action, nut sites. We have selected mutations in the nutR since placement of the n&R region downstream from region that reduce the effectiveness of pN in alter- the bacterial gal operon promoter results in termina- ing transcription initiating at the PR promoter.
    [Show full text]
  • A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes
    Missouri University of Science and Technology Scholars' Mine Computer Science Faculty Research & Creative Works Computer Science 01 Feb 2010 A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes Cyriac Kandoth Fikret Ercaļ Missouri University of Science and Technology, [email protected] Ronald L. Frank Missouri University of Science and Technology, [email protected] Follow this and additional works at: https://scholarsmine.mst.edu/comsci_facwork Part of the Biology Commons, and the Computer Sciences Commons Recommended Citation C. Kandoth et al., "A Framework for Automated Enrichment of Functionally Significant Inverted Repeats in Whole Genomes," BMC Bioinformatics, vol. 11, no. SUPPL. 6, BioMed Central Ltd., Feb 2010. The definitive version is available at https://doi.org/10.1186/1471-2105-11-S6-S20 This Article - Conference proceedings is brought to you for free and open access by Scholars' Mine. It has been accepted for inclusion in Computer Science Faculty Research & Creative Works by an authorized administrator of Scholars' Mine. This work is protected by U. S. Copyright Law. Unauthorized use including reproduction for redistribution requires the permission of the copyright holder. For more information, please contact [email protected]. Kandoth et al. BMC Bioinformatics 2010, 11(Suppl 6):S20 http://www.biomedcentral.com/1471-2105/11/S6/S20 PROCEEDINGS Open Access A framework for automated enrichment of functionally significant inverted repeats in whole genomes Cyriac Kandoth1*, Fikret Ercal1†, Ronald L Frank2† From Seventh Annual MCBIOS Conference. Bioinformatics: Systems, Biology, Informatics and Computation Jonesboro, AR, USA. 19-20 February 2010 Abstract Background: RNA transcripts from genomic sequences showing dyad symmetry typically adopt hairpin-like, cloverleaf, or similar structures that act as recognition sites for proteins.
    [Show full text]
  • DNA Sequence; Operator; Repressor; Regulatory Region; Dyad Symmetry; Plasmid Pbr322; Sl Nuclease Mapping)
    Gene, 23 (1983) 149-156 149 Elsevier Overlapping divergent promoters control expression of TnlO tetracycline resistance (DNA sequence; operator; repressor; regulatory region; dyad symmetry; plasmid pBR322; Sl nuclease mapping) Kevin P. Bertrand *, Kathleen Posh, Lewis V. Wray Jr. ** and William S. Reznikoff ** Department of Microbiology, University of California, Irvine, CA 92717 (U.S.A.) Tel. (714) 833- 6115, and ** Depart- ment of Biochemistry, University of Wisconsin, Madison, WI 53706 (U.S.A.) Tel. (608) 262 - 3608 (Received February 7th, 1983) (Accpeted February l&h, 1983) SUMMARY We have previously examined the genetic organization and regulation of the TnZO tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for retA, the TnlO tetracycline-resistance function, and for tetR, the TnlO lef repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both t&I and t&R is induced by tetracycline. Here we report the DNA sequence of the TnlO tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by Sl nuclease mapping of the 5’ ends of in vivo tet RNA. The t&4 and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the TnlO tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby in~biting transcription initiation at the ietA and tetR promoters. The TnlO ret regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).
    [Show full text]
  • The Regulatory Region of the Divergent Argecbh Operon in Escherichia Coli K-12
    Volume 10 Number 24 1982 Nucleic Acids Research The regulatory region of the divergent argECBH operon in Escherichia coli K-12 Jacques Piette*, Raymond Cunin*, Anne Boyen*, Daniel Charlier*, Marjolaine Crabeel*, Franpoise Van Vliet*, Nicolas Glansdorff*, Craig Squires+ and Catherine L.Squires + *Nficrobiology, Vrije Universiteit Brussel, and Research Institute of the CERIA, 1, Ave. E. Giyson, B-1070 Brussels, Belgium, and +Department of Biological Sciences, Columbia University, New York, NY 10027, USA Received 24 September 1982; Revised and Accepted 24 November 1982 ABSTRACT The nucleotide sequence of the control region of the divergent argECBH operon has been established in the wild type and in mutants affecting expression of these genes. The argE and argCBH promoters face each other and overlap with an operator region containing two domains which may act as distinct repressor binding sites. A long leader sequence - not involved in attenuation - precedes argCBH. Overlapping of the argCBH promoter and the region involved in ribosome mobilization for argE translation explains the dual effect of some mutations. Mutations causing semi-constitutive expression of argE improve putative promoter sequences within argC. Implications of these results regarding control mechanisms in amino acid biosynthesis and their evolution are discussed. INTRODUCTION Divergently transcribed groups of functionally related genes are not exceptional in Escherichia coli (1-10). In only a few instances, however, are the sites responsible for the expression and the regulation of the flanking genes organized in an inte- grated fashion such that the gene cluster constitutes a bipolar operon, with an internal operator region flanked by promoters facing each other. The argECBH cluster (Fig.1) is one of the earliest reported examples of such a pattern.
    [Show full text]
  • A History of CRISPR-CAS
    JB Accepted Manuscript Posted Online 22 January 2018 J. Bacteriol. doi:10.1128/JB.00580-17 Copyright © 2018 American Society for Microbiology. All Rights Reserved. 1 2 3 History of CRISPR-Cas from encounter with a mysterious Downloaded from 4 repeated sequence to genome editing technology 5 6 Yoshizumi Ishino,1, 2,* Mart Krupovic,1 Patrick Forterre1, 3 7 http://jb.asm.org/ 8 1Unité de Biologie Moléculaire du Gène chez les Extrêmophiles, Département de 9 Microbiologie, Institut Pasteur, F-75015, Paris, France, 2Department of 10 Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, on March 9, 2018 by UNIV OF COLORADO 11 Fukuoka 812-8581, Japan. 3Institute of Integrative Cellular Biology, Université 12 Paris Sud, 91405 Orsay, Cedex France 13 14 15 Running title: Discovery and development of CRISPR-Cas research 16 17 * Correspondence to 18 Prof. Yoshizumi Ishino 19 Department of Bioscience and Biotechnology, 20 Faculty of Agriculture, Kyushu University, 21 Fukuoka 812-8581, Japan 22 [email protected] 1 23 ABSTRACT 24 CRISPR-Cas systems are well known acquired immunity systems that are 25 widespread in Archaea and Bacteria. The RNA-guided nucleases from Downloaded from 26 CRISPR-Cas systems are currently regarded as the most reliable tools for 27 genome editing and engineering. The first hint of their existence came in 1987, 28 when an unusual repetitive DNA sequence, which subsequently defined as a 29 cluster of regularly interspersed short palindromic repeats (CRISPR), was http://jb.asm.org/ 30 discovered in the Escherichia coli genome during the analysis of genes involved 31 in phosphate metabolism.
    [Show full text]
  • FARE, a New Family of Foldback Transposons in Arabidopsis
    Copyright 2000 by the Genetics Society of America FARE, a New Family of Foldback Transposons in Arabidopsis Aaron J. Windsor and Candace S. Waddell Department of Biology, McGill University, Montreal, Quebec H3A 1B1, Canada Manuscript received June 23, 2000 Accepted for publication August 16, 2000 ABSTRACT A new family of transposons, FARE, has been identi®ed in Arabidopsis. The structure of these elements is typical of foldback transposons, a distinct subset of mobile DNA elements found in both plants and animals. The ends of FARE elements are long, conserved inverted repeat sequences typically 550 bp in length. These inverted repeats are modular in organization and are predicted to confer extensive secondary structure to the elements. FARE elements are present in high copy number, are heterogeneous in size, and can be divided into two subgroups. FARE1's average 1.1 kb in length and are composed entirely of the long inverted repeats. FARE2's are larger, up to 16.7 kb in length, and contain a large internal region in addition to the inverted repeat ends. The internal region is predicted to encode three proteins, one of which bears homology to a known transposase. FARE1.1 was isolated as an insertion polymorphism between the ecotypes Columbia and Nossen. This, coupled with the presence of 9-bp target-site duplications, strongly suggests that FARE elements have transposed recently. The termini of FARE elements and other foldback transposons are imperfect palindromic sequences, a unique organization that further distinguishes these elements from other mobile DNAs. RANSPOSABLE elements (TEs) are ubiquitous IVR ends and contain no protein coding sequences.
    [Show full text]
  • PG 4Th Semester
    Paper No. : 04 Genetic engineering and recombinant DNA technology Module : 01 History of Genetic Material Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Content Writer: Dr Rohini Muthuswami, Associate Professor, Jawaharlal Nehru University Content Reviwer: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi Genetic Engineering and Recombinant DNA Technology Biotechnology History of Genetic Material Description of Module Subject Name Biotechnology Paper Name Genetic Engineering and Recombinant DNA Technology Module Name/Title History of Genetic Material Module Id 01 Pre-requisites Objectives To understand how DNA was identified as genetic material Keywords DNA Genetic Engineering and Recombinant DNA Technology Biotechnology History of Genetic Material 1. Nature of the genetic material. In this module we will learn about how scientists discovered the identity of the genetic material. The foundation of modern biology was laid by scientists in mid-19th century. The principles of genetics were discovered by Gregor Mendel in 1865-66. The nuclein or what we term as nucleic acid was discovered and identified in 1869. The final identity of the genetic material was firmly established in 1952, two years after the structure of DNA was elucidated. We are going to follow this fascinating journey and I hope at the end of this module you learn to appreciate how scientists using the simple tools and techniques available to them established the identity of the genetic material.
    [Show full text]
  • DNA.Bending by Negative Regulatory Proteins: Gal and Lac Repressors
    Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press DNA.bending by negative regulatory proteins: Gal and Lac repressors Christian Zwieb, Jin Kim, and Sankar Adhya 1 Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 USA The ability of two negative regulatory proteins, Gal and Lac repressors, to induce DNA bending was tested by the respective cloning of ga/and lac operator DNA sequences into a sensitive vector, pBend2, pBend2 can generate a large number of DNA fragments in each of which a cloned operator is present in circularly permuted positions along the length of the DNA. Gel electrophoresis of these DNA fragments individually complexed to a repressor shows that the Gal repressor bends both of the ga/operators, O, and O~. Similarly, the Lac repressor induces a bend to a lac operator DNA. In each case, the center of the average bent segment is located at or close to the dyad symmetry axis of the operator sequence. In view of these findings, we discuss how these negative regulatory proteins may function by a more dynamic mechanism than was perceived previously. [Key Words: DNA bending; Gal and Lac repressors; gal and lac operators] Received January 13, 1989; revised version accepted March 6, 1989. It is becoming increasingly clear that biological macro- Parker 1986; Dripps and Wartell 1987; Mizuno 1987; molecular reactions involve the formation of specific Plaskon and Wartell 1987). It was suggested by Crothers DNA-multiprotein complexes. For many of these reac- and Fried (1983)that such contortions may be essential tions, such as replication or recombination, the com- for the formation and activity of the DNA-protein tran- plexes assume a higher-order structure (Echols 1986}.
    [Show full text]
  • A Protein Involved in Minichromosome Maintenance in Yeast Binds a Transcriptional Enhancer Conserved in Eukaryotes
    Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes Steven Passmore, Randolph Elble, and Bik-Kwoon Tye Section of Biochemistry, Molecular and Cell Biology, Comell University, Ithaca, New York 14853 USA The Saccharomyces cerevisiae MCM1 gene product is a protein with multiple functions. It is a transcription factor necessary for expression of mating-type-specific genes and is also required for the maintenance of minichromosomes. MCM1 shows DNA-binding specificities similar to those of two previously reported DNA- binding factors, pheromone/receptor transcription factor (PRTF) and general regulator of mating type (GRM)~ like PRTF, its activity can be modulated by the al protein. MCM1 binds to the dyad symmetry element 5'- CCTAATTAGG and related sequences, which we refer to as MCM1 _control _elements (MCEs). MCEs are found within the regulatory regions of a- and ,,-specific genes. Direct and indirect DNA binding assays suggest that a conserved 5'-ATTAGG in one-half of the dyad symmetry element is important for MCM1 binding whereas variants in the other half are tolerated. We have used a novel DNase I 'nicking interference' assay to investigate the interaction of MCM1 with its substrate. These data suggest that MCM1 binds as a dimer, interacting symmetrically with the ATTAGG residues in each half of the binding site. MCM1 contains striking homology to the DNA-binding domain of the human serum response factor (SRF) which mediates the transient transcriptional activation of growth-stimulated genes by binding to the serum response element (SRE).
    [Show full text]
  • Analysis of the DNA-Binding and Activation Properties of the Human Transcription Factor AP-2
    Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Analysis of the DNA-binding and activation properties of the human transcription factor AP-2 Trevor Williams ~ and Robert Tjian Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720 USA The mammalian transcription factor AP-2 is a sequence-specific DNA-binding protein expressed in neural crest lineages and regulated by retinoic acid. Here we report a structure/function analysis of the DNA-binding and transcription activation properties of the AP-2 protein. DNA contact studies indicate that AP-2 binds as a dimer to a palindromic recognition sequence. Furthermore, cross-linking and immunoprecipitation data illustrate that AP-2 exists as a dimer even in the absence of DNA. Examination of cDNA mutants reveals that the sequences responsible for DNA binding are located in the carboxy-terminal half of the protein. In addition, a domain mediating dimerization forms an integral component of this DNA-binding structure. Expression of AP-2 in mammalian cells demonstrates that transcriptional activation requires an additional amino-terminal domain that contains an unusually high concentration of proline residues. This proline-rich activation domain also functions when attached to the heterologous DNA-binding region of the GAL4 protein. This study reveals that although AP-2 shares an underlying modular organization with other transcription factors, the regions of AP-2 involved in transcriptional activation and DNA binding/dimerization have novel sequence characteristics. [Key Words: Enhancer factor~ DNA binding~ dimerization; mammalian expression~ proline-rich activator~ palindromic binding site] Received November 15, 19901 revised version accepted January 22, 1991.
    [Show full text]
  • Escherichia Coli Σ38 Promoters Use Two up Elements Instead of a −35 Element: Resolution of a Paradox and Discovery That Σ38 Transcribes Ribosomal Promoters
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.05.936344; this version posted February 6, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license. Escherichia coli σ38 promoters use two UP elements instead of a −35 element: resolution of a paradox and discovery that σ38 transcribes ribosomal promoters Kevin S. Franco†,1, Zhe Sun†,1, Yixiong Chen1, Cedric Cagliero2, Yuhong Zuo3, Yan Ning Zhou1, Mikhail Kashlev1, Ding Jun Jin1 and Thomas D. Schneider 1,∗ version = 3.15 of sigma38.tex 2020 Jan 08 1National Institutes of Health, National Cancer Institute, Center for Cancer Research, RNA Biology Laboratory, P. O. Box B, Frederick, MD 21702-1201. (301) 846-5581; [email protected]; https://alum.mit.edu/www/toms/. 2 National Institutes of Health, National Cancer Institute, Center for Cancer Research, Gene Regulation and Chromosome Bi- ology Laboratory, P. O. Box B, Frederick, MD 21702-1201. Jecho Laboratories, Inc. 7320A Executive way, Frederick, MD 21704. 3Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA. † These authors contributed equally to this work. ∗Corresponding author. 1 Abstract In E. coli, one RNA polymerase (RNAP) transcribes all RNA species, and different regu- lons are transcribed by employing different sigma (σ) factors. RNAP containing σ38 (σS ) activates genes responding to stress conditions such as stationary phase. The structure of σ38 promoters has been controversial for more than two decades.
    [Show full text]
  • III = Chpt. 17. Transcriptional Regulation in Bacteriophage Lambda
    B M B 400 Part Four - III = Chpt. 17. Transcriptional regulation in bacteriophage lambda B M B 400 Part Four: Gene Regulation Section III = Chapter 17 TRANSCRIPTIONAL REGULATION IN BACTERIOPHAGE LAMBDA Not all bacteriophage lyse their host bacteria upon infection. Temperate phage reside in the host genome and do not kill the host, whereas lytic phage cause lysis of their hosts when they infect bacteria. The bacteriophage λ can choose between these two “lifestyles.” The molecular basis for this decision is one of the best understood genetic switches that has been studied, and it provides a fundamental paradigm for such molecular switches in developmental biology. This chapter reviews some of the historical observations on lysogeny in bacteriophage λ, covers the major events in lysis and lysogeny, and discusses the principal regulatory proteins and their competition for overlapping cis-regulatory sites. We will examine one of the common DNA binding domains in regulatory proteins – the helix-turn-helix, which was first identified in the λ Cro protein. Also, the use of hybrid genes to dissect complex regulatory schemes was pioneered in studies of bacteriophage λ, and that approach will be discussed in this chapter. A. Lysis versus lysogeny 1. Lytic pathway: a. Leads to many progeny virus particles and lysis of the infected cell. b. Have extensive replication of λ DNA, formation of the viral coat (head and tail proteins) with packaging of the λ DNA into phage particles, cell lysis, and release of many progeny phage. 2. Lysogenic pathway: a. The infecting phage DNA integrates into the host genome and is carried passively by the host.
    [Show full text]