RESUMO
Mitochondrial carnitine palmitoyltransferases I and II (CPTI and CPTII), together with the carnitine carrier, transport long-chain fatty acyl-CoA from the cytosol to the mitochondrial matrix for beta-oxidation. Recent progress in the expression of CPTI and CPTII cDNA clones in Pichia pastoris, a yeast with no endogenous CPT activity, has greatly facilitated the characterization of these important enzymes in fatty acid oxidation. It is now well established that yeast-expressed CPTI is a catalytically active, malonyl CoA-sensitive, distinct enzyme that is reversibly inactivated by detergents. CPTII is a catalytically active, malonyl CoA-insensitive, distinct enzyme that is detergent stable. Reconstitution studies with yeast-expressed CPTI have established for the first time that detergent inactivation of CPTI is reversible, suggesting that CPTI is active only in a membrane environment. By constructing a series of deletion mutants of the N-terminus of liver CPTI, we have mapped the residues essential for malonyl CoA inhibition and binding to the conserved first six N-terminal amino acid residues. Mutation of glutamic acid 3 to alanine abolished malonyl CoA inhibition and high affinity malonyl CoA binding, but not catalytic activity, whereas mutation of histidine 5 to alanine caused partial loss in malonyl CoA inhibition. Our mutagenesis studies demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl CoA inhibition and binding to liver CPTI, but not catalytic activity.
Assuntos
Carnitina O-Palmitoiltransferase/fisiologia , Ácidos Graxos/metabolismo , Malonil Coenzima A/antagonistas & inibidores , Mitocôndrias/enzimologia , Pichia/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carnitina O-Palmitoiltransferase/metabolismo , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Pichia/metabolismoRESUMO
Heart/skeletal muscle carnitine palmitoyltransferase I (M-CPTI) is 30-100-fold more sensitive to malonyl CoA inhibition than the liver isoform (L-CPTI). To determine the role of the N-terminal region of human heart M-CPTI on malonyl CoA sensitivity and binding, a series of deletion mutations were constructed ranging in size from 18 to 83 N-terminal residues. All of the deletions except Delta83 were active. Mitochondria from the yeast strains expressing Delta28 and Delta39 exhibited a 2.5-fold higher activity compared to the wild type, but were insensitive to malonyl CoA inhibition and had complete loss of high-affinity malonyl CoA binding. The high-affinity site (K(D1), B(max1)) for binding of malonyl CoA to M-CPTI was completely abolished in the Delta28, Delta39, Delta51, and Delta72 mutants, suggesting that the decrease in malonyl CoA sensitivity observed in these mutants was due to the loss of the high-affinity binding entity of the enzyme. Delta18 showed only a 4-fold loss in malonyl CoA sensitivity but had activity and high-affinity malonyl CoA binding similar to the wild type. Replacement of the N-terminal domain of L-CPTI with the N-terminal domain of M-CPTI does not change the malonyl CoA sensitivity of the chimeric L-CPTI, suggesting that the amino acid residues responsible for the differing sensitivity to malonyl CoA are not located in this N-terminal region. These results demonstrate that the N-terminal residues critical for activity and malonyl CoA sensitivity in M-CPTI are different from those of L-CPTI.
Assuntos
Aminoácidos/fisiologia , Carnitina O-Palmitoiltransferase/metabolismo , Malonil Coenzima A/metabolismo , Miocárdio/enzimologia , Fragmentos de Peptídeos/fisiologia , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Sítios de Ligação/genética , Radioisótopos de Carbono , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/biossíntese , Carnitina O-Palmitoiltransferase/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Vetores Genéticos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Malonil Coenzima A/farmacologia , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Músculo Esquelético/metabolismo , Mutagênese Sítio-Dirigida , Miocárdio/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Pichia/genética , Ratos , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
We have recently shown by deletion mutation analysis that the conserved first 18 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) are essential for malonyl-CoA inhibition and binding (Shi, J., Zhu, H., Arvidson, D. N. , Cregg, J. M., and Woldegiorgis, G. (1998) Biochemistry 37, 11033-11038). To identify specific residue(s) involved in malonyl-CoA binding and inhibition of L-CPTI, we constructed two more deletion mutants, Delta12 and Delta6, and three substitution mutations within the conserved first six amino acid residues. Mutant L-CPTI, lacking either the first six N-terminal amino acid residues or with a change of glutamic acid 3 to alanine, was expressed at steady-state levels similar to wild type and had near wild type catalytic activity. However, malonyl-CoA inhibition of these mutant enzymes was reduced 100-fold, and high affinity malonyl-CoA binding was lost. A mutant L-CPTI with a change of histidine 5 to alanine caused only partial loss of malonyl-CoA inhibition, whereas a mutant L-CPTI with a change of glutamine 6 to alanine had wild type properties. These results demonstrate that glutamic acid 3 and histidine 5 are necessary for malonyl-CoA binding and inhibition of L-CPTI by malonyl-CoA but are not required for catalysis.
Assuntos
Alanina/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Ácido Glutâmico/metabolismo , Fígado/enzimologia , Malonil Coenzima A/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Western Blotting , Malonil Coenzima A/metabolismo , Dados de Sequência Molecular , Mutação Puntual , Ratos , Relação Estrutura-AtividadeRESUMO
To assess the role of the 130 N-terminal amino acid residues of rat liver carnitine palmitoyltransferase I (L-CPTI) on malonyl-CoA sensitivity and binding, we constructed a series of mutants with deletions of the 18, 35, 52, 73, 83, or 129 most N-terminal amino acid residues. The deletion mutants were expressed in the yeast Pichia pastoris. We determined the effects of these mutations on L-CPTI activity, malonyl-CoA sensitivity, and binding in isolated mitochondria prepared from the yeast strains expressing the wild-type and deletion mutants. The mutant protein that lacked the first 18 N-terminal amino acid residues, Delta18, had activity and kinetic properties similar to wild-type L-CPTI, but it was almost completely insensitive to malonyl-CoA inhibition (I50 = 380 microM versus 2.0 microM). In addition, loss of malonyl-CoA sensitivity in Delta18 was accompanied by a 70-fold decrease in affinity for malonyl CoA (KD = 70 nM versus 1.1 nM) compared to wild-type L-CPTI. Deletion of the first 35, 52, 73, and 83 N-terminal amino acid residues had a similar effect on malonyl-CoA sensitivity as did the 18-residue deletion mutant, and there was a progressive reduction in the affinity for malonyl-CoA binding. By contrast, deletion of the first 129 N-terminal amino acid residues resulted in the synthesis of an inactive protein. To our knowledge, this is the first report to demonstrate a critical role for these perfectly conserved first 18 N-terminal amino acid residues of L-CPTI in malonyl-CoA sensitivity and binding.
Assuntos
Carnitina O-Palmitoiltransferase/genética , Sequência Conservada/genética , Fígado/enzimologia , Malonil Coenzima A/metabolismo , Fragmentos de Peptídeos/genética , Deleção de Sequência , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Radioisótopos de Carbono , Carnitina O-Palmitoiltransferase/biossíntese , Carnitina O-Palmitoiltransferase/metabolismo , Ativação Enzimática/genética , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Pichia/enzimologia , Pichia/genética , Plasmídeos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The crystal structure of the purine repressor mutant L54M bound to hypoxanthine and to the purF operator provides a stereochemical understanding of the high DNA affinity of this hinge helix mutant. Comparison of the PurR L54M-DNA complex to that of the wild type PurR-DNA complex reveals that these purine repressors bind and kink DNA similarly despite significant differences in their minor groove contacts and routes to interdigitation of the central C.G:G.C base pair step. Modeling studies, supported by genetic and biochemical data, show that the stereochemistry of the backbone atoms of the abutting hinge helices combined with the rigidity of the kinked base pair step constrain the interdigitating residue to leucine or methionine for the LacI/GalR family of transcription regulators.
Assuntos
Substituição de Aminoácidos/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Conformação de Ácido Nucleico , Proteínas Repressoras/química , Proteínas Repressoras/genética , Cristalografia por Raios X , Leucina/genética , Metionina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Guanine or hypoxanthine, physiological corepressors of the Escherichia coli purine repressor (PurR), promote formation of the ternary PurR-corepressor-operator DNA complex that functions to repress pur operon gene expression. Structure-based predictions on the importance of Arg190 in determining 6-oxopurine specificity and corepressor binding affinity were tested by mutagenesis, analysis of in vivo function, and in vitro corepressor binding measurements. Replacements of Arg190 with Ala or Gln resulted in functional repressors in which binding of guanine and hypoxanthine was retained but specificity was relaxed to permit binding of adenine. X-ray structures were determined for ternary complexes of mutant repressors with purines (adenine, guanine, hypoxanthine, and 6-methylpurine) and operator DNA. These structures indicate that R190A binds guanine, hypoxanthine, and adenine with nearly equal, albeit reduced, affinity in large part because of a newly made compensatory hydrogen bond between the rotated hydroxyl side chain of Ser124 and the exocyclic 6 positions of the purines. Through direct and water-mediated contacts, the R190Q protein binds adenine with a nearly 75-fold higher affinity than the wild type repressor while maintaining wild type affinity for guanine and hypoxanthine. The results establish at the atomic level the basis for the critical role of Arg190 in the recognition of the exocyclic 6 position of its purine corepressors and the successful redesign of corepressor specificity.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Purinas/metabolismo , Proteínas Repressoras/genética , Adenina/metabolismo , Alanina/genética , Arginina/genética , Cristalografia por Raios X , Repressão Enzimática , Glutamina/genética , Guanina/metabolismo , Hipoxantina/metabolismo , Ligantes , Mutagênese Sítio-Dirigida , Conformação Proteica , Engenharia de ProteínasRESUMO
Long-chain fatty acids are the primary source of energy production in the heart. Carnitine palmitoyltransferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in beta-oxidation. In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeast Pichia pastoris. Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone. The clone has an open reading frame of 2316 bp with a 5' untranslated region of 38 bp and a 256-bp 3' untranslated region with the poly(A)+ addition sequence AATAAA. The predicted protein has 772 amino acids and a molecular mass of 88 kDa. Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of approximately 3.1 kb that was only present in human heart and skeletal muscle tissue. Expression of the human M-CPT-I cDNA in P. pastoris, a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria. Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile. The I50 for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nM, and the Kms for carnitine and palmitoyl-CoA were 666 and 42 microM, respectively. The I50 for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and the Km for carnitine is more than 20 times higher than that of the liver CPT-I. This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Miocárdio/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Carnitina/metabolismo , Carnitina O-Palmitoiltransferase/análise , Carnitina O-Palmitoiltransferase/genética , Clonagem Molecular , Expressão Gênica , Humanos , Immunoblotting , Cinética , Malonil Coenzima A/farmacologia , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Palmitoil Coenzima A/metabolismo , Pichia/enzimologia , Pichia/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismoRESUMO
The rate-limiting step in beta oxidation is the conversion of long-chain acyl-CoA to acylcarnitine, a reaction catalyzed by the outer mitochondrial membrane enzyme carnitine palmitoyltransferase I (CPTI) and inhibited by malonyl-CoA. The acylcarnitine is then translocated across the inner mitochondrial membrane by the carnitine/acylcarnitine translocase and converted back to acyl-CoA by CPTII. Although CPTII has been examined in detail, studies on CPTI have been hampered by an inability to purify CPTI in an active form from CPTII. In particular, it has not been conclusively demonstrated that CPTI is even catalytically active, or whether sensitivity of CPTI to malonyl-CoA is an intrinsic property of the enzyme or is contained in a separate regulatory subunit that interacts with CPTI. To address these questions, the genes for CPTI and CPTII were separately expressed in Pichia pastoris, a yeast with no endogenous CPT activity. High levels of CPT activity were present in purified mitochondrial preparations from both CPTI- and CPTII-expressing strains. Furthermore, CPTI activity was highly sensitive to inhibition by malonyl-CoA while CPTII was not. Thus, CPT catalytic activity and malonyl-CoA sensitivity are contained within a single CPTI polypeptide in mammalian mitochondrial membranes. We describe the kinetic characteristics for the yeast-expressed CPTs, the first such report for a CPTI enzyme in the absence of CPTII. Yeast-expressed CPTI is inactivated by detergent solubilization. However, removal of the detergent in the presence of phospholipids resulted in the recovery of malonyl-CoA-sensitive CPTI activity, suggesting that CPTI requires a membranous environment. CPTI is thus reversibly inactivated by detergents.
Assuntos
Carnitina O-Palmitoiltransferase/genética , Acil Coenzima A/metabolismo , Animais , Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/metabolismo , DNA Complementar/química , DNA Complementar/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Cinética , Lipossomos , Fígado/enzimologia , Malonil Coenzima A/metabolismo , Dados de Sequência Molecular , Pichia , RatosRESUMO
Two different X-ray co-crystal structures of the Escherichia coli trp holorepressor complexed with DNA suggest that the TrpR protein recognizes specific DNA sequences primarily with a network of water-mediated H-bonds. However, the more recent nuclear magnetic resonance (NMR) solution structures of the holorepressor-operator complex show no long-lived, ordered water molecules at the protein-DNA interface and place amino acids in intimate contact with nucleotide bases. Both genetic and biochemical studies support a model in which the trp repressor recognizes specific DNA sequences by a direct mechanism, as seen in the NMR solution structures, not by the 'indirect readout' mechanism initially proposed on the basis of X-ray studies.
Assuntos
Proteínas de Bactérias , DNA Bacteriano/metabolismo , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Triptofano , Sequência de Bases , DNA Bacteriano/química , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas Repressoras/químicaRESUMO
Site-directed mutagenesis was used to construct mutant Trp repressors with each of the 38 possible single amino acid changes of the first 2 amino acid residues (Ile79 and Ala80) in the second "recognition" alpha-helix of the helix-turn-helix DNA-binding motif. Eight of these mutant repressors with Ile79 and Ala80 changes are more active than the wild-type protein when tryptophan is limiting, and are super-aporepressors. Eleven mutant repressors have extended DNA-binding specificies in vivo, and bind operators which the wild-type repressor cannot. One mutant repressor, Lys79, has a classical altered specificity phenotype in vivo, and binds the wild-type trp operator less well than wild-type repressor, yet binds a mutant operator better than wild-type repressor. A site-specific nuclease was derived from Lys79 repressor by constructing a double-mutant protein with Lys79 and a sole cysteine residue, Cys49, and alkylating this cysteine with a 1,10-phenanthroline-copper adduct. This nuclease has an altered specificity of DNA binding in vitro. When activated by the addition of thiol and hydrogen peroxide, the Lys79 nuclease cleaves operator DNA within its new recognition sequence with high efficiency.
Assuntos
Proteínas de Bactérias , DNA/química , DNA/metabolismo , Desoxirribonucleases/metabolismo , Estrutura Secundária de Proteína , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Bases , Sítios de Ligação , Desoxirribonucleases/química , Escherichia coli/genética , Escherichia coli/metabolismo , Sequências Hélice-Alça-Hélice , Cinética , Modelos Estruturais , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Especificidade por SubstratoRESUMO
Threonine is found at the third position of the second alpha-helix in the helix-turn-helix motifs of most bacterial DNA-binding proteins. To investigate the role of this conserved residue in Escherichia coli Trp repressor function, plasmids encoding mutant Trp repressors with each of the 19 amino acid changes of Thr-81 were made by site-directed mutagenesis. All 19 changes decrease the activity of Trp holorepressor, indicating that the Thr-81 side-chain is critical for TrpR function. Three mutant repressors, Ser-81, Lys-81 and Arg-81, retain partial DNA-binding activity and inhibit transcription from the wild-type trp promoter/operator complex; challenge-phage assays show that Ser-81 and Lys-81 holorepressors have altered DNA-binding specificities. The side-chain of Thr-81 may make direct contacts with base pairs 4 and 3 of the trp operator, consistent with the nuclear magnetic resonance solution structures of the holorepressor-operator complex.
Assuntos
Proteínas de Bactérias , DNA Bacteriano/metabolismo , Escherichia coli/genética , Sequências Hélice-Alça-Hélice , Proteínas Repressoras/genética , Treonina/química , Sequência de Aminoácidos , Apoproteínas/metabolismo , Sequência de Bases , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Regiões Operadoras Genéticas , Ligação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Repressoras/metabolismo , Salmonella typhimurium/metabolismo , Relação Estrutura-AtividadeRESUMO
Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.
Assuntos
Proteínas de Bactérias , Sequência Conservada , Enterobacteriaceae/genética , Genes Bacterianos/genética , Proteínas Repressoras/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/análise , Enterobacter/genética , Escherichia coli/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Óperon/genética , Filogenia , Conformação Proteica , Sequências Reguladoras de Ácido Nucleico , Salmonella typhimurium/genética , Alinhamento de Sequência , Deleção de Sequência/genéticaRESUMO
The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors. However, none of the four mutant repressors binds DNA in a corepressor-independent manner. Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro. Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins. These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Escherichia coli/genética , Mutação Puntual , Ligação ProteicaRESUMO
The binding of L-tryptophan to Escherichia coli tryptophan aporepressor enables the holorepressor complex to bind operator DNA tightly. The side chain of residue alanine 77 is located in one of the most flexible regions of Trp repressor, between residues critical for binding DNA. Codon-directed mutagenesis was used to make genes encoding mutant Trp repressors with each of the 19 naturally occurring amino acid changes of Ala77. The 19 mutant proteins are made at the same steady-state levels as wild type. Sensitive challenge phage assays show that 7 of the 19 mutant proteins (Cys, Ser, Val, Leu, Thr, Ile, and Lys) are more active than wild-type protein when tryptophan is limiting in vivo. Among these 7 mutant super-aporepressors, proteins with Cys and Ser changes also are super-holorepressors, because they repress better than wild-type holorepressor when tryptophan is in excess. These results and others suggest that super-aporepressors associate more poorly than wild-type aporepressor with nonspecific DNA. Consistent with this idea, these 7 changes are predicted to disrupt the tertiary structure of aporepressor, but have more limited effects on the structure of holorepressor.
Assuntos
Alanina/metabolismo , Apoproteínas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Triptofano/metabolismo , Alanina/genética , Sequência de Aminoácidos , Apoproteínas/genética , Proteínas de Bactérias , Sequência de Bases , DNA Bacteriano/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Repressoras/genética , Triptofano/genéticaRESUMO
The binding of the corepressor, L-tryptophan, to the Escherichia coli trp-aporepressor in solution has been examined by 13C- and 19F-NMR spectroscopy. The binding of a number of tryptophan analogues have been studied by equilibrium dialysis. Evidence is presented that support the crystallographic studies (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L. and Sigler, P. B. (1985) Nature 317, 782-786) that Val-58 is within the ring currents of the bound tryptophan and also close in space to the indole 5'-position, on the basis of heteronuclear 19F(1H)-NOE experiments. The tryptophan carboxylate is in hydrogen-bonding distance to a highly positively charged residue, probably Arg-54 and this bond strengthens on formation of the trp-repressor-DNA complex.
Assuntos
Núcleo Celular/química , Proteínas Repressoras/química , Escherichia coli , Espectroscopia de Ressonância Magnética , Triptofano/análogos & derivados , ValinaRESUMO
An automated kinetic assay for beta-galactosidase activity in Escherichia coli was developed to permit the measurement of many independent samples simultaneously. Bacteria are grown, lysed from without (by adsorption of a high multiplicity of bacteriophage T4) and assayed in microtiter plates with 96 wells. Absorbance data are collected and analyzed by computer. The growth and lysis procedure, apparatus and software used in this assay can be used for other spectrophotometric enzyme assays.
Assuntos
Escherichia coli/enzimologia , beta-Galactosidase/análise , Automação , Cinética , Espectrofotometria UltravioletaRESUMO
The Escherichia coli trpR gene encodes tryptophan aporepressor, which binds the corepressor ligand, L-tryptophan, to form an active repressor complex. The side chain of residue valine 58 of Trp aporepressor sits at the bottom of the corepressor (L-tryptophan) binding pocket. Mutant trpR genes encoding changes of Val58 to the other 19 naturally occurring amino acids were made. Each of the mutant proteins requires a higher intracellular concentration of tryptophan for activation of DNA binding than wild-type aporepressor. Whereas wild-type aporepressor is activated better by 5-methyltryptophan (5-MT) than by tryptophan, Ile58 and other mutant aporepressors prefer tryptophan to 5-MT as corepressor, and Ala58 and Gly58 prefer 5-MT much more strongly than wild-type aporepressor in vivo. These mutant aporepressors are the first examples of DNA-binding proteins with altered specificities of cofactor recognition.
Assuntos
Apoproteínas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Mutação , Proteínas Repressoras/genética , Triptofano/metabolismo , Apoproteínas/metabolismo , Proteínas de Bactérias , Sequência de Bases , Regulação da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Repressoras/metabolismo , Valina/químicaRESUMO
We have analyzed the sequence-specific interaction between the Escherichia coli tryptophan (Trp) repressor and its operator using challenge phage vectors. These phages, derivatives of Salmonella phage P22 that have substitutions of synthetic, symmetric trp operators for the P22 mnt operator, provide a genetic assay for DNA binding in vivo. Phages carrying operators that retain the determinants of Trp repressor binding efficiently lysogenize cells producing repressor; in contrast, phages with operators missing critical determinants kill such hosts. The binding determinants revealed by this assay corroborate a simple docking model for the Trp repressor-operator interaction postulated from the repressor crystal structure, and account for both the specificity of repressor binding and the ability of Trp repressor to recognize multiple, tandem DNA sites.
Assuntos
Proteínas de Bactérias , Escherichia coli/genética , Óperon , Plasmídeos , Proteínas Repressoras/metabolismo , Fagos de Salmonella/genética , Salmonella typhimurium/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , DNA Bacteriano/metabolismo , Ligação ProteicaRESUMO
We have examined the interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan, using both equilibrium dialysis and flow dialysis methods. Results obtained by the two procedures were equivalent and indicate that the trp aporepressor binds L-tryptophan with an equilibrium dissociation constant (Kd) of 40 microM at 25 degrees C under standard binding assay conditions (10 mM potassium phosphate, pH 7.4, 0.2 M potassium chloride, 0.1 mM EDTA, 5% glycerol). Molecular sizing of the purified trp aporepressor shows that in the absence of ligand the regulatory protein exists as a dimeric species with greater than 99% purity and an apparent molecular weight of 30,000. Under the storage and assay conditions used, the dimer appears quite stable, and essentially no monomer or higher multimeric species are detected. Analysis of binding data by Scatchard and direct linear plot methods shows two identical and independent ligand-binding sites/native trp aporepressor dimer. When examined as a function of temperature, L-tryptophan binding by trp aporepressor varied over 7-fold (Kd = 28 microM at 6.5 degrees C to Kd = 217 microM at 40 degrees C). At the optimal growth temperature for E. coli (37 degrees C), the dissociation constant was 160 microM for the ligand, L-tryptophan. From the relationship between temperature and L-tryptophan binding by trp aporepressor, the apparent enthalpy change delta H = -10.6 +/- 0.6 kcal mol-1 and the apparent entropy change delta S = -17 +/- 2 cal degree-1 mol-1 were determined.
Assuntos
Escherichia coli/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Triptofano/metabolismo , Cromatografia Líquida de Alta Pressão , Glutaral/metabolismo , Cinética , Substâncias Macromoleculares , Peso Molecular , TemperaturaRESUMO
A single-base deletion within the protein-coding region of the adenovirus type 5 early region 1A (E1A) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an E1A protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress E1A mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of E1A which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated E1A protein does not produce the cis-acting defect in E1A transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 E1A. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for E1A transcription during the late phase of infection.