Página 1 dos resultados de 2796 itens digitais encontrados em 0.016 segundos

‣ Glucose and glucose-6-phosphate interaction with Xyl repressor proteins from Bacillus spp. may contribute to regulation of xylose utilization.

Dahl, M K; Schmiedel, D; Hillen, W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1995 Português
Relevância na Pesquisa
47.022563%
The xyl operons of several gram-positive bacteria are regulated at the level of transcription by xylose-responsive repressor proteins (XylR). In addition, they are catabolite repressed. Here, we describe a mechanism by which glucose metabolism can affect both regulatory mechanisms. Glucose-6-phosphate appeared to be an anti-inducer of xyl operon transcription, since it could compete with xylose in interaction in vitro with XylR from Bacillus subtilis, B. megaterium, and B. licheniformis. On the other hand, glucose was a low-efficiency inactivator of XylR from B. subtilis and B. megaterium and a weak anti-inducer of XylR from B. licheniformis. Thus, the chemical nature of the substituent at C-5 of xylose and the primary structure of XylR determine the effect of these compounds on xyl operon transcription.

‣ The bovine papillomavirus type 1 E2 transactivator and repressor proteins use different nuclear localization signals.

Skiadopoulos, M H; McBride, A A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1996 Português
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The E2 gene of bovine papillomavirus type 1 encodes at least three nuclear phosphoproteins that regulate viral transcription and DNA replication. All three proteins have a common C-terminal domain that has DNA-binding and dimerization activities. A basic region in this domain forms an alpha helix which makes direct contact with the DNA target. In this study, it is shown that in addition to its role in DNA binding, this basic region functions as a nuclear localization signal both in the E2 DNA-binding domain and in a heterologous protein. Deletion of this signal sequence resulted in increased accumulation of the E2 transactivator and repressor proteins in the cytoplasm, but nuclear localization was not eliminated. In the full-length transactivator protein, another signal, present in the N-terminal transactivation domain, is used for transport to the nucleus, and the C-terminal nuclear localization signal(s) are masked. The use of different nuclear localization signals could potentially allow differential regulation of the subcellular localization of the E2 transactivator and repressor proteins at some stage in the viral life cycle.

‣ MalI, a novel protein involved in regulation of the maltose system of Escherichia coli, is highly homologous to the repressor proteins GalR, CytR, and LacI.

Reidl, J; Römisch, K; Ehrmann, M; Boos, W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1989 Português
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The maltose regulon of Escherichia coli comprises several operons that are under common regulatory control of the MalT activator protein. Five mal genes, organized in two divergent operons, code for a binding-protein-dependent transport system specific for maltose and maltodextrins. MalK, one of the subunits of this transport system, not only is essential for transport but also plays a role in regulation. Mutations abolishing MalK function not only result in inability to transport maltose but also cause constitutive expression of the maltose regulon. For this constitutivity to be exerted, the function of an additional gene product, MalI, is necessary. Using the constitutive expression of a malK-lacZ fusion as a signal, we cloned the malI gene, expressed it in minicells, and determined its DNA sequence. The sequence predicted a protein of 34,729 molecular weight, in agreement with the apparent molecular weight of the protein (35,000) when expressed in minicells and analyzed by polyacrylamide gel electrophoresis and autoradiography. MalI exhibited high homology to the repressor proteins GalR, CytR, and LacI. When the amino acid sequences were appropriately aligned, MalI showed 28% identity to GalR, 21% to CytR, and 24% to LacI. Including conservative amino acid exchanges...

‣ The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins.

Heinrich, J; Riedel, H D; Baumstark, B R; Kimura, M; Schuster, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/10/1989 Português
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47.77076%
The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. A rapid purification procedure was required for the isolation of the thermolabile repressor proteins. Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins. Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor. Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA. This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.

‣ Structure of the DNA-binding region of lac repressor inferred from its homology with cro repressor.

Matthews, B W; Ohlendorf, D H; Anderson, W F; Takeda, Y
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1982 Português
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37.822383%
It is shown that the amino acid sequence and the DNA gene sequence of the 25 amino-terminal residues of the lac repressor protein of Escherichia coli are homologous with the sequences of five DNA-binding proteins: the cro repressor proteins from phage lambda and phage 434, the cI and cII proteins from phage lambda, and the repressor protein from Salmonella phage P22. The region of homology between lac repressor and the other proteins coincides with the principal DNA-binding region of cro repressor. In particular, residues Tyr-17 through Gln-26 of lac repressor correspond to the alpha-helix Gln-27 through Ala-36 of cro repressor, which we have postulated to bind within the major groove of the DNA and to be primarily responsible for the recognition of the DNA operator region by the protein [Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754--758]. By analogy with cro repressor, we propose that residues 17--26 of lac repressor are alpha-helical and that this helix and a twofold-related alpha-helix in an adjacent subunit bind within successive major grooves of the lac operator, which is in a right-handed Watson--Crick B-DNA conformation. Also, by analogy with cro repressor, we suggest that residues Thr-5 through Ala-13 of lac repressor form a second alpha-helix and contribute...

‣ Structural similarity in the DNA-binding domains of catabolite gene activator and cro repressor proteins.

Steitz, T A; Ohlendorf, D H; McKay, D B; Anderson, W F; Matthews, B W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1982 Português
Relevância na Pesquisa
47.4437%
It is shown that there is a structural similarity between the presumed DNA-binding regions of the Escherichia coli catabolite gene activator protein ("CAP") and the cro repressor protein ("cro") from bacteriophage lambda. The correspondence between the two proteins is particularly striking for a structural unit consisting of two consecutive alpha-helices. The 24 alpha-carbon atoms that constitute the two-helical structural units in the two proteins can be superimposed with a root-mean-square disagreement of 1.1 A. It is shown that this agreement is very unlikely to be due to a chance correspondence. For both CAP activator and cro repressor proteins it is the second alpha-helix of the two-helical unit that has been proposed to bind within the major groove of left-handed or right-handed B DNA, respectively [McKay, D. B. & Steitz, T. A. (1981) Nature (London) 290, 744-749; Anderson, W. F., Ohlendorf, D. H., Takeda, Y. & Matthews, B. W. (1981) Nature (London) 290, 754-758]. The structural correspondence between CAP and cro seen here, together with other recent evidence of sequence homologies between cro, CAP, and other proteins that bind double-stranded DNA, suggests that the two-helical unit is likely to be a common feature of many DNA-binding proteins. The results also suggest that some principles of specific protein-double-stranded DNA interaction may be general and include recognition via alpha-helices fitting into the major groove of the DNA.

‣ Induction of Vascular Smooth Muscle α-Actin Gene Transcription in Transforming Growth Factor β1-Activated Myofibroblasts Mediated by Dynamic Interplay between the Pur Repressor Proteins and Sp1/Smad Coactivators

Subramanian, Sukanya V.; Polikandriotis, John A.; Kelm, Robert J.; David, Jason J.; Orosz, Charles G.; Strauch, Arthur R.
Fonte: The American Society for Cell Biology Publicador: The American Society for Cell Biology
Tipo: Artigo de Revista Científica
Publicado em /10/2004 Português
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47.4437%
The mouse vascular smooth muscle α-actin (SMA) gene enhancer is activated in fibroblasts by transforming growth factor β1 (TGFβ1), a potent mediator of myofibroblast differentiation and wound healing. The SMA enhancer contains tandem sites for the Sp1 transcriptional activator protein and Purα and β repressor proteins. We have examined dynamic interplay between these divergent proteins to identify checkpoints for possible control of myofibroblast differentiation during chronic inflammatory disease. A novel element in the SMA enhancer named SPUR was responsible for both basal and TGFβ1-dependent transcriptional activation in fibroblasts and capable of binding Sp1 and Pur proteins. A novel Sp1:Pur:SPUR complex was dissociated when SMA enhancer activity was increased by TGFβ1 or Smad protein overexpression. Physical association of Pur proteins with Smad2/3 was observed as was binding of Smads to an upstream enhancer region that undergoes DNA duplex unwinding in TGFβ1-activated myofibroblasts. Purβ repression of the SMA enhancer could not be relieved by TGFβ1, whereas repression mediated by Purα was partially rescued by TGFβ1 or overexpression of Smad proteins. Interplay between Pur repressor isoforms and Sp1 and Smad coactivators may regulate SMA enhancer output in TGFβ1-activated myofibroblasts during episodes of wound repair and tissue remodeling.

‣ Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and ribosome association.

Muckenthaler, M; Gunkel, N; Stripecke, R; Hentze, M W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1997 Português
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The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.

‣ WD40 Domain Divergence Is Important for Functional Differences between the Fission Yeast Tup11 and Tup12 Co-Repressor Proteins

Ferreira, Monica E.; Berndt, Kurt D.; Nilsson, Johan; Wright, Anthony P. H.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 08/06/2010 Português
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47.224062%
We have previously demonstrated that subsets of Ssn6/Tup target genes have distinct requirements for the Schizosaccharomyces pombe homologs of the Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very high level of divergence in the histone interacting repression domains of the two proteins suggested that determinants distinguishing Tup11 and Tup12 might be located in this domain. Here we have combined phylogenetic and structural analysis as well as phenotypic characterization, under stress conditions that specifically require Tup12, to identify and characterize the domains involved in Tup12-specific action. The results indicate that divergence in the repression domain is not generally relevant for Tup12-specific function. Instead, we show that the more highly conserved C-terminal WD40 repeat domain of Tup12 is important for Tup12-specific function. Surface amino acid residues specific for the WD40 repeat domain of Tup12 proteins in different fission yeasts are clustered in blade 3 of the propeller-like structure that is characteristic of WD40 repeat domains. The Tup11 and Tup12 proteins in fission yeasts thus provide an excellent model system for studying the functional divergence of WD40 repeat domains.

‣ Cooperativity in long-range gene regulation by the λ CI repressor; Cooperativity in long-range gene regulation by the lambda CI repressor

Dodd, I.B.; Shearwin, K.E.; Perkins, A.J.; Burr, T.; Hochschild, A.; Egan, J.B.
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em //2004 Português
Relevância na Pesquisa
47.625566%
Effective repression of cI transcription from PRM by the bacteriophage λ CI repressor requires binding sites (OL) located 2.4 kb from the promoter. A CI tetramer bound to OL1.OL2 interacts with a tetramer bound near PRM (OR1.OR2), looping the intervening DNA. We previously proposed that in this CI octamer:DNA complex, the distant OL3 operator and the weak OR3 operator overlapping PRM are juxtaposed so that a CI dimer at OL3 can cooperate with a CI dimer binding to OR3. Here we show that OL3 is necessary for effective repression of PRM and that the repressor at OL3 appears to interact specifically with the repressor at OR3. The OL3-CI-OR3 interaction involves the same CI interface used for short-range dimer-dimer interactions and does not occur without the other four operators. The long-range interactions were incorporated into a physicochemical model, allowing estimation of the long-range interaction energies and showing the lysogenic state to be ideally poised for CI negative autoregulation. The results establish the λ system as a powerful tool for examining long-range gene regulatory interactions in vivo.; Ian B. Dodd, Keith E. Shearwin, Alison J. Perkins, Tom Burr, Ann Hochschild, and J. Barry Egan

‣ Action at a distance in Cl repressor regulation of the bacteriophage 186 genetic switch

Dodd, I.; Egan, J.
Fonte: Blackwell Science Ltd Publicador: Blackwell Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2002 Português
Relevância na Pesquisa
47.37252%
The non-lambdoid coliphage 186 provides an alternative model to the lytic-lysogenic switch of phage λ. Like λ, the key switch regulator, the CI repressor, associates to octamers. Unlike λ, the lytic promoter (pR) and the lysogenic promoter (pL) are face-to-face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located ≈ 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to λ. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co-operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.; Ian B. Dodd and J. Barry Egan

‣ Role of RegM, a homologue of the catabolite repressor protein CcpA, in the virulence of Streptococcus pneumoniae

Giammarinaro, P.; Paton, J.
Fonte: Amer Soc Microbiology Publicador: Amer Soc Microbiology
Tipo: Artigo de Revista Científica
Publicado em //2002 Português
Relevância na Pesquisa
47.4437%
As part of a study of virulence gene regulation in Streptococcus pneumoniae, we have identified a gene encoding a homologue of the staphylococcal catabolite control protein CcpA in the pneumococcal genome sequence. The pneumococcal protein, designated RegM, has significant similarity to members of the LacI/GalR family of bacterial regulatory proteins. S. pneumoniae D39 derivatives with insertion-duplication or deletion mutations in regM were significantly attenuated in virulence with respect to the wild-type strain. In defined media containing either sucrose or lactose as sole carbon sources, the in vitro growth rates of D39 and the regM mutants were essentially the same. However, in the presence of galactose the regM mutants grew significantly faster than the wild-type strain, whereas growth rates were significantly lower in the presence of glucose or maltose. These data are consistent with the involvement of regM in the catabolism of carbohydrates in S. pneumoniae. RegM was a repressor of both -glucosidase and ß-galactosidase activities in S. pneumoniae, but unlike the situation in certain other bacteria, it does not mediate the repression of these enzymes by glucose. The observed attenuation in virulence was not attributable to poorer growth of the regM mutants in mouse blood ex vivo...

‣ Aristaless-related homeobox gene, the gene responsible for west syndrome and related disorders, is a groucho/transducin-like enhancer of split dependent transcriptional repressor

McKenzie, O.; Ponte, I.; Mangelsdorf, M.; Finnis, M.; Colasante, G.; Shoubridge, C.; Stifani, S.; Gecz, J.; Broccoli, V.
Fonte: Pergamon-Elsevier Science Ltd Publicador: Pergamon-Elsevier Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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47.56843%
Aristaless-related homeobox gene (ARX) is an important paired-type homeobox gene involved in the development of human brain. The ARX gene mutations are a significant contributor to various forms of X-chromosome-linked mental retardation with and without additional features including epilepsy, lissencephaly with abnormal genitalia, hand dystonia or autism. Here we demonstrate that the human ARX protein is a potent transcriptional repressor, which binds to Groucho/transducin-like enhancer of split (TLE) co-factor proteins and the TLE1 in particular through its octapeptide (Engrailed homology repressor domain (eh-1) homology) domain. We show that the transcription repression activity of ARX is modulated by two strong repression domains, one located within the octapeptide domain and the second in the region of the polyalanine tract 4, and one activator domain, the aristaless domain. Importantly, we show that the transcription repression activity of ARX is affected by various naturally occurring mutations. The introduction of the c.98T>C (p.L33P) mutation results in the lack of binding to TLE1 protein and relaxed transcription repression. The introduction of the two most frequent ARX polyalanine tract expansion mutations increases the repression activity in a manner dependent on the number of extra alanines. Interestingly...

‣ Characterization of ankyrin repeat-containing proteins as substrates of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible transcription factor

Karttunen, S.; Hampton-Smith, R.; Peet, D.
Fonte: Elsevier Academic Press Inc Publicador: Elsevier Academic Press Inc
Tipo: Artigo de Revista Científica
Publicado em //2007 Português
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46.967446%
The hypoxia-inducible transcription factors (HIFs) are essential mediators of the genomic response to oxygen deficiency (hypoxia) in multicellular organisms. The HIFs are regulated by four oxygen-sensitive hydroxylases-three prolyl hydroxylases and one asparaginyl hydroxylase. These hydroxylases are all members of the 2-oxoglutarate (2OG)-dependent dioxygenase superfamily and convey changes in cellular oxygen concentration to the HIF-alpha (alpha) subunit, leading to potent accumulation and activity in hypoxia versus degradation and repression in normoxia. HIF-alpha asparaginyl hydroxylation is catalyzed by factor-inhibiting HIF-1 (FIH-1) and directly regulates the transcription activity of the HIF-alpha proteins. Recent work has demonstrated that, in addition to hydroxylating HIF-alpha, FIH-1 can also hydroxylate the ankyrin domains of a wide range of proteins. This paper presents in vitro and cell-based techniques for the preliminary characterization of ankyrin domain-containing proteins as FIH-1 substrates and interacting proteins. Strategies are presented for the expression and purification of FIH-1 from mammalian or bacterial cells. Similar to the HIF-alpha proteins, the ankyrin-containing substrates are examined as purified proteins expressed in bacteria and overexpressed in mammalian cells or in the form of synthetic peptides. Specific conditions for the efficient expression of ankyrin-containing proteins compared with the HIF-alpha substrates in Escherichia coli are detailed. Hydroxylation is rapidly inferred...

‣ Nedd4 Family-interacting Protein 1 (Ndfip1) Is Required for the Exosomal Secretion of Nedd4 Family Proteins

Putz, U.; Howitt, J.; Lackovic, J.; Foot, N.; Kumar, S.; Silke, J.; Tan, S.
Fonte: Amer Soc Biochemistry Molecular Biology Inc Publicador: Amer Soc Biochemistry Molecular Biology Inc
Tipo: Artigo de Revista Científica
Publicado em //2008 Português
Relevância na Pesquisa
46.98283%
The ability to remove unwanted proteins is an important cellular feature. Classically, this involves the enzymatic addition of ubiquitin moieties followed by degradation in the proteasome. Nedd4 proteins are ubiquitin ligases important not only for protein degradation, but also for protein trafficking. Nedd4 proteins can bind to target proteins either by themselves or through adaptor protein Ndfip1 (Nedd4 family-interacting protein 1). An alternative mechanism for protein removal and trafficking is provided by exosomes, which are small vesicles (50–90-nm diameter) originating from late endosomes and multivesicular bodies (MVBs). Exosomes provide a rapid means of shedding obsolete proteins and also for cell to cell communication. In the present work, we show that Ndfip1 is detectable in exosomes secreted from transfected cells and also from primary neurons. Compared with control, Ndfip1 increases exosome secretion from transfected cells. Furthermore, while Nedd4, Nedd4-2, and Itch are normally absent from exosomes, expression of Ndfip1 results in recruitment of all three Nedd4 proteins into exosomes. Together, these results suggest that Ndfip1 is important for protein trafficking via exosomes, and provides a mechanism for cargoing passenger proteins such as Nedd4 family proteins. Given the positive roles of Ndfip1/Nedd4 in improving neuronal survival during brain injury...

‣ Single molecule analysis of DNA wrapping and looping by a circular 14mer wheel of the bacteriophage 186 CI repressor

Wang, H.; Dodd, I.; Dunlap, D.; Shearwin, K.; Finzi, L.
Fonte: Oxford Univ Press Publicador: Oxford Univ Press
Tipo: Artigo de Revista Científica
Publicado em //2013 Português
Relevância na Pesquisa
47.329146%
The lytic–lysogenic decision in bacteriophage 186 is governed by the 186 CI repressor protein in a unique way. The 186 CI is proposed to form a wheel-like oligomer that can mediate either wrapped or looped nucleoprotein complexes to provide the cooperative and competitive interactions needed for regulation. Although consistent with structural, biochemical and gene expression data, many aspects of this model are based on inference. Here, we use atomic force microscopy (AFM) to reveal the various predicted wrapped and looped species, and new ones, for CI regulation of lytic and lysogenic transcription. Automated AFM analysis showed CI particles of the predicted dimensions on the DNA, with CI multimerization favoured by DNA binding. Measurement of the length of the wrapped DNA segments indicated that CI may move on the DNA, wrapping or releasing DNA on either side of the wheel. Tethered particle motion experiments were consistent with wrapping and looping of DNA by CI in solution, where in contrast to λ repressor, the looped species were exceptionally stable. The CI regulatory system provides an intriguing comparison with that of nucleosomes, which share the ability to wrap and release similar sized segments of DNA.; Haowei Wang, Ian B. Dodd...

‣ Control of gene expression in the temperate coliphage 186. X. The cl repressor directly represses transcription of the late control gene B

Dibbens, J.A.; Gregory, S.L.; Egan, J.B.
Fonte: Wiley Publicador: Wiley
Tipo: Artigo de Revista Científica
Publicado em //1992 Português
Relevância na Pesquisa
47.40826%
We have found that the repressor of 186 lytic transcription, CI, represses transcription of the late control gene B, with no involvement of the B protein Itself. In clone studies we showed that CI repressed transcription from the B promoter and that temperature inactivation of CIts led to B derepression. We conclude that CI repressor directly represses transcription of the Bgene and, with prophage induction, it is probable that the inactivation of the CI repressor not only derepresses early lytic transcription, but also derepresses B gene transcription, leading to the activation of transcription from the late promoters.; Justin A. Dibbens, Stephen L. Gregory and J. Barry Egan

‣ Structurial studies of thelac repressor using homology modeling and combinatorial mutations

Nichols, Jeffry Curtis
Fonte: Universidade Rice Publicador: Universidade Rice
Português
Relevância na Pesquisa
37.796338%
The core domain (residues 62-323) of the regulatory protein lac repressor has been aligned to several sugar binding proteins of known structure. The overall homology based on two separate matrix scoring systems (minimum base change per codon and amino acid homology per residue) is significant. Similarly, the predicted secondary structure of the repressor exhibits excellent agreement with the known secondary structures of the sugar binding proteins. Using this primary sequence alignment, the tertiary structure of the core domain of lac repressor was modeled using the structures of the sugar binding proteins as templates. Further refinements of this model using the purine repressor provided improvement in regions not well defined based on the sugar binding proteins. Important residues involved in operator and sugar binding and in protein assembly have been identified using genetic methods, and placement of these residues in the model is consistent with their known function. The recent solution of the crystallographic structure confirms the elements of this homology model. This approach provides an effective means to visualize the core domain of the lac repressor and to interpret the mutational data for specific residues. The availability of models of this type provides a structural basis for rational design of experiments. Mutations that affect both monomer-monomer and dimer-dimer subunit interfaces have been combined to generate a new family of mutant proteins designed to explore the role of subunit interactions in this regulatory protein. Combination of apolar substitutions at residue 84 and C-terminal deletions to generate dimer results in mutant repressor proteins with increased stability...

‣ Repressor proteins: Ligand binding, thermodynamics and assembly

Xu, Han
Fonte: Universidade Rice Publicador: Universidade Rice
Português
Relevância na Pesquisa
47.18273%
LacI and PurR are members of the extended LacI/GalR family of bacterial proteins that regulate genetic expression. Detailed kinetic and thermodynamic studies on PurR were compared with results for homologous LacI. Operator binding affinity was increased by the presence of guanine as demonstrated previously, and conversely guanine binding affinity was increased by the presence of operator. Guanine enhanced operator affinity by increasing the association rate constant and decreasing the dissociation rate constant for binding. Operator had minimal effect on the association rate constant for guanine binding; however, this DNA decreased the dissociation rate constant for corepressor by ∼10-fold. Despite significant sequence and structural similarity between PurR and LacI proteins, PurR binds to its corepressor ligand with a lower association rate constant than LacI binds to its inducer ligand. However, the rate constant for PurR-guanine binding to operator is ∼3-fold higher than for LacI binding to its cognate operator under the same solution conditions. The distinct metabolic roles of the enzymes under regulation by these two repressor proteins provide a rationale for the observed functional differences. Addition of the LacI C-terminal tetramerization domain to the C-terminus of PurR resulted in the tetramerization of PurR. This tetrameric mutant of PurR exhibited very similar corepressor and operator binding affinity to wild-type PurR. These results establish that the C-terminal assembly motif from LacI can elicit tetramer formation in a naturally folded homologous dimer without interference with its biological function. Studies of the N-terminal DNA binding domain of LacI with variant hinge regions linked by a disulfide bond confirmed that the hinge region is important for operator recognition and interaction. DNA binding domains containing the hinge region from LacI (LH and VH) have similar affinity to lacO 1 operator...

‣ Lactose repressor proteins with increased operator DNA binding affinity

Matthews, Kathleen S.; Foster, Catherine M.; Swint-kruse, Liskin
Fonte: Universidade Rice Publicador: Universidade Rice
Português
Relevância na Pesquisa
47.56843%
The present invention provides altered lac repressor proteins that recognize the lactose operator with increased affinity and have either normal or enhanced ligand responsivity. For example, the lac repressor Gln60Gly mutant protein exhibits increased binding affinity for lactose operator DNA, while maintaining near-normal responsivity to IPTG. Alternatively, the present invention provides modified repressors which exhibit responsiveness to an alternative ligand, such as arabinose, or have enhanced responsivity to IPTG. For example, Gln60Gly/Leu148Phe binds with wild-type affinity to lactose operator DNA and exhibits enhanced responsivity to IPTG. The present invention also provides for repressors that exhibit both characteristics: increased affinity for lactose operator and enhanced ligand responsivity. Enhanced ligand response enables induction of gene expression to be finely controlled by a researcher. DNA sequences encoding the altered lac repressor proteins and bacterial and eukaryotic cells containing altered lac repressor proteins are also provided.