Assuntos
Biofilmes/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Vibrio cholerae/genética , Vibrio cholerae/fisiologia , DNA Complementar/genética , Genoma Bacteriano , Humanos , Fases de Leitura Aberta , Fenótipo , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Bacteriano/isolamento & purificação , Vibrio cholerae/classificação , Vibrio cholerae/patogenicidadeRESUMO
At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dimer, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of CRP is responsible for the transcriptional inactivity of the complexes.
Assuntos
Proteína Receptora de AMP Cíclico/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica , Sequência de Bases , Escherichia coli , Proteínas de Escherichia coli , Modelos Moleculares , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Conformação ProteicaRESUMO
The Escherichia coli cAMP receptor protein (CRP) is essential for the activation and repression of transcription initiation at promoters in the CytR regulon. CRP performs these activities by making direct protein-protein interactions to the alpha-subunits of RNA polymerase and to the CytR regulator. Strikingly, it has been shown that amino acids of CRP that are critical for communication with the two partner proteins are located in close proximity on the surface of CRP. Here, we have dissected this surface in order to pinpoint the 'repression region' of CRP and to assess whether it overlaps with the characterized 'activating region'. Our results established that residues 12, 13, 17, 105, 108 and 110 are essential for the interaction with CytR and confirmed that 'activating region' 2 of CRP is made up of residues 19, 21 and 101. In the crystallographic structure of the CRP-DNA complex, the two sets of determinants are located immediately adjacent to each other forming a consecutive surface-exposed patch. The 'repression region' is chemically complementary to the characterized region on CytR that is essential for protein-protein communication to CRP. Moreover, the results provide insight into the mechanism by which CytR might prevent CRP-mediated transcription.
Assuntos
Proteínas de Bactérias/química , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas Repressoras/química , Alanina , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte , Proteína Receptora de AMP Cíclico/genética , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas Repressoras/metabolismo , Ativação TranscricionalRESUMO
The present investigation examined the possible influence of urinary calcium excretion on the concentration of renal calbindin-D28k. Thiazide diuretics stimulate calcium transport across the epithelial cells of the distal tubule, which express calbindin-D28k in high concentrations. Calbindin-D28k is assumed to facilitate transcellular Ca diffusion. Reduced urine calcium excretion and increased urine output were induced in Wistar rats by infusion of bendroflume-thiazide 1 mg/kg/day. The two control groups had infusions of either furosemide 20 mg/kg/day or vehicle, n = 8 in each group. Urinary Ca excretion was reduced to 10% in the thiazide group and increased by 50% in the furosemide group. Renal concentrations of calbindin-D28 showed no difference between vehicle, thiazide- and furosemide-treated rats. No differences in plasma concentrations of calcium, magnesium, phosphorus, urea, PTH, calcitonin and 1,25-(OH)2D were found between the groups. The present study describes that urine calcium excretion selectively can be manipulated without accompanying changes in renal calbindin-D28k concentrations. The data, therefore, suggest that urinary calcium excretion is not a significant determinator of cytosolic concentrations of renal calbindin-D28k.