REGULATION OF PROTEIN SYNTHESIS: AN ALTERNATIVE TO THE REPRESSOR-OPERATOR HYPOTHESIS* M. GRUBER and R. N. CAMPAGNE\ ... by specific "effectors," i.e., inducers and "co-repressors." These substances—usually compounds of low molecular weight— ... immediate derivatives, were assumed to interact with a "repressor," preventing the transcription ofthe operon into RNA. The stereospecific interaction with the effector would change the repressor by an allosteric transition [3], thus inducing or ... preventing or allowing the transcription ofthe operon. The repressor was assumed to be the product of a ...
Tópico(s): Protein Structure and Dynamics
1966 - Johns Hopkins University Press | Perspectives in biology and medicine
Louis F. Reichardt, A.D. Kaiser,
Direct measurements of the intracellular level of λ repressor have been made by a DNA-filter assay ... assay. Transcription of cI , the structural gene for repressor, appears to initiate at two different promoters, prm ... cY do not make a normal burst of repressor after infection and do not efficiently lysogenize the cell. Cro product stops repressor synthesis midway in the infective cycle. Promoter prm maintains the repressor level in established lysogens. Delection mapping places it ... the right operator ( Or ). Prm is activated by repressor bound to the right operator. In the absence ...
Tópico(s): Immune Response and Inflammation
1971 - National Academy of Sciences | Proceedings of the National Academy of Sciences
Alan H. Jobe, Arthur D. Riggs, Suzanne Bourgeois,
... for measuring inducer and operator binding of lac repressor in crude extract. For most experiments, including those measuring repressor-operator association rates, purification beyond ammonium sulfate fractionation ... required. Kinetic measurements have been used to differentiate repressors of three normally inducible strains. Repressor from strain C6000 binds operator more tightly than does repressor from E203, previously considered to be wild type and subjected to extensive analysis in this laboratory. Repressor from an overproducing (iq) strain is also different ...
Tópico(s): Enzyme Structure and Function
1972 - Elsevier BV | Journal of Molecular Biology
Ray Wü, Chander P. Bahl, Saran A. Narang,
This chapter discusses the lactose operator–repressor interaction. Any method for quantitatively studying the operator–repressor interaction requires either the operator or the repressor species to be labeled. After the interaction has ... methods have been used to study the operator–repressor interaction. Gilbert and Muller-Hill used 35S-labeled lac repressor. The operator–repressor complex was separated from the unreacted lac repressor by zonal centrifugation on a 5–30% glycerol ... Bourgeois for studying various properties of the operator–repressor complex. In this method, the DNA–protein complex ...
Tópico(s): DNA and Nucleic Acid Chemistry
1978 - Academic Press | Current topics in cellular regulation
Ying Kao-Huang, Arnold Revzin, Andrew P. Butler, Pamela O'conner, Daniel W. Noble, Peter H. von Hippel,
... estimate of the fraction of Escherichia coli lac repressor molecules bound in vivo to the operator region ... is derived by measurement of the distribution of repressor between a minicell-producing E. coli strain (P678- ... we find that less than 10% of the repressor tetramers of the average cell are free in ... in vivo value of the association constant for repressor to bulk nonoperator DNA (K(RD)) calculated from ... the association constant for the binding of inducer-repressor complex to bulk nonoperator DNA (K(RID)) is [ ...
Tópico(s): RNA and protein synthesis mechanisms
1977 - National Academy of Sciences | Proceedings of the National Academy of Sciences
David V. Goeddel, Daniel G. Yansura, Marvin H. Caruthers,
... wild-type (SQ) and tight-binding (QX86) lac repressors with synthetic lac operators 21 and 26 base pairs long. The repressor binding properties of both operators were very similar, indicating that both contain the same specific repressor recognition sites. The repressor-operator association rate constants ( k a ) were more ... natural conditions there are electrostatic interactions between lac repressor and DNA regions outside of the 26 base pair operator sequence. Association rate constants for SQ repressor with either operator are higher than have been ...
Tópico(s): Advanced biosensing and bioanalysis techniques
1977 - National Academy of Sciences | Proceedings of the National Academy of Sciences
Jürgen Kania, Benno Müller‐Hill,
... which produce hybrid molecules between the chimaeric protein repressor-galactosidase and the enzyme beta-galactosidase, were constructed. Repressor-galactosidase in which fully active lac repressor is covalently linked to active beta-galactosidase, is ... four beta-galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric beta-galactosidase, retain all the biological activities of tetrameric lac repressor. Substitution of repressor-galactosidase subunits with beta-galactosidase ...
Tópico(s): Amino Acid Enzymes and Metabolism
1977 - Wiley | European Journal of Biochemistry
Amy C. Wang, Arnold Revzin, Andrew P. Butler, Peter H. von Hippel,
... is shown by melting profile analysis of lac repressor-DNA complexes that repressor binds tightly and preferentially (relative to single-stranded ... binding stabilizes the DNA against melting and the repressor against thermal denaturation. Analysis of the extent of stabilization and the rate of dissociation of repressor from non-operator DNA as a function of ... 10) M(-1) at 0.002 M Na(+). Repressor bound to non-operator DNA is not further ... used to measure the rate of dissociation of repressor from operator DNA. These experiments show that most ...
Tópico(s): Bacterial Genetics and Biotechnology
1977 - Oxford University Press | Nucleic Acids Research
Timothy J. Richmond, Thomas A. Steitz,
From studies of repressor binding to poly[d(A · U-HgX)], a structural analog of poly[d(A · T)], we demonstrate that the lac repressor does not make extensive contacts in the major ... chains. These modified DNAs bind well to the repressor (Kdis = 10−7 to 10−9 m). Thus, the high affinity of repressor for these non-operator DNA molecules cannot involve ... major groove. These studies make those models for repressor-DNA interaction which involve binding of an α- ... groove of the DNA or binding of the repressor to cruciform or Gierer-like structures (1966) not ...
Tópico(s): Bacteriophages and microbial interactions
1976 - Elsevier BV | Journal of Molecular Biology
In vitro measurements show that the X86 repressor, which has an increased affinity for the lac operator as compared to wild-type repressor, also has an increased affinity for non-operator ... coli DNA. The rate constant of association of repressor and operator is decreased by E. coli DNA fivefold more for X86 repressor than for wild-type repressor. Low inducer concentrations increase the rate of association of X86 repressor and operator in the presence of E. coli ... partial equilibrium situation where part of the X86 repressor is bound to the operator, and part to ...
Tópico(s): RNA Interference and Gene Delivery
1976 - Elsevier BV | Journal of Molecular Biology
Felicia Y.-H. Wu, Pradip K. Bandyopadhyay, Cheng-Wen Wu,
Temperature-jump studies of the lac repressor were performed monitoring the intrinsic tryptophan fluorescence of the protein. A single relaxation process with a time constant of 1·36×104 s−1 was observed for lac repressor solutions at pH 7·5 in the absence ... time is independent of the concentration of lac repressor indicating a conformational transition between two states of the repressor (R*гR). Although the binding of an inducer, ... interconversion between these two states of the lac repressor, the relaxation amplitude decreases with increasing IPTG† concentration. ...
Tópico(s): Protein Structure and Dynamics
1976 - Elsevier BV | Journal of Molecular Biology
The chimaeric protein repressor-galactosidase, in which fully active lac repressor is covalently linked to the active enzyme beta-galactosidase, was used as ... system for probing the quaternary structure of lac repressor. Electron micrographs revealed repressor-galactosidase to be a tetrameric aggregate. When lac repressor, alone, was crosslinked with dimethyl suberimidate, dimers, trimers, ... subunit were produced, whereas crosslinking of the tetrameric repressor-galactosidase resulted in the production of only dimers ...
Tópico(s): Amino Acid Enzymes and Metabolism
1976 - National Academy of Sciences | Proceedings of the National Academy of Sciences
David C. Hagen, Boris Magasanik,
... and hut(P,R,Q)UH. A single repressor, the product of the C gene, regulates both ... The deoxyribonucleic acid (DNA)-binding activity of the repressor was measured using DNA's containing separate operators. The repressor had greater activity when assayed using DNA containing ... binding activities were also determined for two super-repressors. The super-repressors had altered DNA-binding properties, although the self-regulated nature of the repressors complicated the analysis of the results. A purfication ...
Tópico(s): Bacterial Genetics and Biotechnology
1976 - American Society for Microbiology | Journal of Bacteriology
... this promoter. cro product does not inactivate λ repressor, the cI gene product, and cro product also acts effectively in the presence of active repressor. Therefore, cro product seems to block directly the ... a site which can not be protected by repressor binding to the right operator Or. Since cro ... which it blocks cI. My experiments indicate that repressor must be bound to the right operator Or ... expression from prm is not constitutive. Pre-existing repressor stimulates sevenfold the initial rate of active repressor ...
Tópico(s): Escherichia coli research studies
1975 - Elsevier BV | Journal of Molecular Biology
Treatment of Escherichia coli lac repressor with iodine (Kl3) at 4 degrees and pH 7.5 resulted in the rapid loss of repressor DNA binding activity. At a 30-fold molar excess of iodine to repressor, inactivation was complete within 15 sec. Inducer binding ... the same conditions was only slightly affected. Iodinated repressor remained tetrameric, indicating that no gross structural alteration ... restoration of binding activity was observed when iodinated repressor was assayed under a variety of assay conditions. ...
Tópico(s): Amino Acid Enzymes and Metabolism
1975 - American Chemical Society | Biochemistry
Christine Brack, Vincenzo Pirrotta,
The structure of purified phage λ repressor has been examined by high resolution electron microscopy. The repressor molecule appears predominantly as a tetramer of about 95 Å × 120 Å. ... of the DNA and the structure of the repressor molecule bound to it. Mapping the positions of λ repressor bound to whole λ DNA shows preferential binding ... the operators. At high resolution multiple binding of repressor to the operator can be demonstrated. Depending on the amount of repressor present, rows of one to four repressor tetramers ...
Tópico(s): Monoclonal and Polyclonal Antibodies Research
1975 - Elsevier BV | Journal of Molecular Biology
Miriam M. Susskind, David Botstein,
... the synthesis or activity of the P22 c2 repressor. In this paper, we show that P22 anti-repressor interferes with phage λ cI repressor (which is analogous in function to P22 c2 repressor), since superinfection of a λ lysogen with P22 results in dis-appearance of λ repressor activity. Antirepressor interferes directly with repressor activity rather than indirectly with repressor synthesis, since antirepressor in crude P22-infected cell extracts inhibits λ repressor activity in vitro. Under our assay conditions, antirepressor ...
Tópico(s): Bacterial Genetics and Biotechnology
1975 - Elsevier BV | Journal of Molecular Biology
Peter Y. Chou, Alice J. Adler, Gerald D. Fasman,
... Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence ... α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for ... 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while ... sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two ... for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were ... 324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “ ...
Tópico(s): Carbohydrate Chemistry and Synthesis
1975 - Elsevier BV | Journal of Molecular Biology
... subsample of 38) carried mutations in the lac repressor gene. There were two classes of repressor mutants. As well as the commonly identified class of mutants with completely inactivated repressors, there was a frequent class of mutants (21/37) whose repressors were partially inactivated. Most of these (15/21) ... to levels characteristic of the parent strain. The repressor activities were diverse and stably expressed under routine ... not result from the formation of temperature-sensitive repressors. None of the mutants with completely inactivated repressors ...
Tópico(s): DNA and Nucleic Acid Chemistry
1974 - American Society for Microbiology | Journal of Bacteriology
Ronald B. O'Gorman, Marietta Dunaway, Kathleen S. Matthews,
The nonspecific DNA binding capacity of repressor protein has been assessed by boundary sedimentation of repressor and calf thymus DNA fragmented by shearing and by nitrocellulose ultrafiltration employing labeled ... of inducer concentrations sufficient to insure dissociation of repressor from the operator region of the DNA. These ... literature. The association constants for the interaction of repressor with operator DNA fragments and lambdaplac DNA have ... physiological value. The equilibrium association constant for the repressor-operator DNA fragment is significantly less sensitive to ...
Tópico(s): Prion Diseases and Protein Misfolding
1980 - Elsevier BV | Journal of Biological Chemistry
Ronald B. O'Gorman, John M. Rosenberg, Olga B. Kallai, Richard E. Dickerson, Keiichi Itakura, Arthur D. Riggs, Kathleen S. Matthews,
The characteristics of inducer binding to lactose repressor protein in the presence of a small (29 base pair) operator DNA fragment have been examined. The presence of operator DNA fragments decreases ... the presence of operator DNA fragments, while free repressor binds inducer in a noncooperative fashion. The experimental data for the binding of inducer to repressor both in the presence and absence of saturating ... the limits placed by the measured values for repressor-operator DNA-inducer interaction, it was possible to ...
Tópico(s): RNA Interference and Gene Delivery
1980 - Elsevier BV | Journal of Biological Chemistry
Barbara J Meyer, Russell Maurer, Mark Ptashne,
... two regulatory proteins. These proteins are the λ repressor, the product of the cI gene, and the ... PRM in vivo as a function of λ repressor or cro protein concentration. To accomplish this we ... λ regulatory protein is controlled by the lac repressor. Cells bearing one of these plasmids synthesize increased amounts of λ repressor or cro in response to increased amounts of ... PRM functions efficiently only in the presence of repressor, prmup-1 allows initiation to occur at PRM at high frequency in the absence of repressor. Our main results may be summarized as follows. ( ...
Tópico(s): Protein purification and stability
1980 - Elsevier BV | Journal of Molecular Biology
Shigetada Nakanishi, Sankar Adhya, Max E. Gottesman, Ira Pastan,
... repression of gal mRNA synthesis by the gal repressor from Escherichia coli. By use of a four- ... DEAE-cellulose, and an affinity resin, the gal repressor has been purified about 1600-fold from a ... lysogenic for bacteriophage lambda that carries the gal repressor gene (galR). The highly purified gal repressor is an effective and specific repressor of in vitro synthesis of gal mRNA with ... and D-galactose overcome the action of gal repressor; the half-maximal concentrations of D-fucose and D-galactose for overcoming the action of repressor are 1 mM and 0.5 mM, respectively. ...
Tópico(s): Biopolymer Synthesis and Applications
1973 - National Academy of Sciences | Proceedings of the National Academy of Sciences
Alain Lévine, Adriana Bailone, Raymond Devoret,
... a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels. Using the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to ... without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied ...
Tópico(s): Protein purification and stability
1979 - Elsevier BV | Journal of Molecular Biology
Adriana Bailone, Alain Lévine, Raymond Devoret,
... induction results from the inactivation of the λ repressor by a cellular inducer. Although it has been shown that the phage A repressor is inactivated by the recA gene product in ... action of the cellular inducer on the λ repressor, we made use of bacteria with elevated cellular levels of the λ repressor (hyperimmune lysogens). We determined the kinetics of repressor inactivation promoted by three representative inducing treatments: ultraviolet ... up of tif-1 mutants. The kinetics of repressor decay in wild-type monolysogens indicate that repressor ...
Tópico(s): Bacteriophages and microbial interactions
1979 - Elsevier BV | Journal of Molecular Biology
Manfred SCHLOTMANN, Konrad Beyreuther,
... has been shown that 28 transdominant mutant lac repressors which have lost operator DNA-binding ability in ... and are able to form tetramers (i-d repressors), could be divided into two groups by their ... All but one of 15 analysed i-d repressors with amino acid substitutions to the C-terminal ... specific DNA binding. All 13 tested i-d repressors with amino acid substitutions to the N-terminal ... to double-stranded DNA, and 11 of these repressors derived from missense mutations in the lacI gene ... residues producing a mutant-specific pattern of stable repressor fragments. These fragments are tetrameric and capable of ...
Tópico(s): Bacterial Genetics and Biotechnology
1979 - Wiley | European Journal of Biochemistry
Alexander D. Johnson, Barbara J Meyer, Mark Ptashne,
The λ phage repressor binds cooperatively to the three sites in the right operator ( O R ) according to the following pattern. If the ... 2 are filled coordinately because of interactions between repressor dimers bound to these two sites. Site O R 3 is filled only at higher repressor concentrations. In contrast, if O R 1 is ... 3 are filled coordinately because of interactions between repressors bound to these sites. In this case, the ... to the wild type. We infer that a repressor dimer bound to the middle site O R ...
Tópico(s): RNA Interference and Gene Delivery
1979 - National Academy of Sciences | Proceedings of the National Academy of Sciences
Pradip K. Bandyopadhyay, Cheng-Wen Wu,
The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, ... isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions ... fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ1 = 5.3 ...
Tópico(s): Mass Spectrometry Techniques and Applications
1979 - Elsevier BV | Archives of Biochemistry and Biophysics
Robert T. Sauer, Michael J. Ross, Mark Ptashne,
... recA cleavage of the phage X and P22 repressors has been determined.Each repressor is cut once by the recA enzyme, X repressor between residues 111 and 112, and P22 repressor between residues 94 and 95.recA cleavage occurs at identical alanyl-glycyl sequences in both repressors, and in both repressors, the cleavage separates the repressor's NHz-terminal DNA-binding domain from its ... domain.A papain-generated proteolytic fragment of X repressor consisting of repressor residues 93-236 is also efficiently cleaved by ...
Tópico(s): Genomics and Chromatin Dynamics
1982 - Elsevier BV | Journal of Biological Chemistry
Anthony R. Poteete, Mark Ptashne,
Bacteriophage P22 repressor regulates transcription from two divergent promoters, PRand PRM, by binding to three sites in the right operator of the phage. Repressor binding depends on the intrinsic affinities of the ... on pairwise co-operative interactions between adjacently bound repressors. In the case of wild-type OR, repressor binds with the affinity order: OR1 − OR2 > OR3. With repressor bound to OR1 and OR2, the early lytic ... in part, from direct stimulation of transcription by repressor bound to OR2. At high concentrations, repressor binds ...
Tópico(s): Protein Structure and Dynamics
1982 - Elsevier BV | Journal of Molecular Biology