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Titolo:
ASSOCIATION STATES OF THE TRANSCRIPTION ACTIVATOR PROTEIN NTRC FROM ESCHERICHIA-COLI DETERMINED BY ANALYTICAL ULTRACENTRIFUGATION
Autore:
RIPPE K; MUCKE N; SCHULZ A;
Indirizzi:
DEUTSCH KREBSFORSCHUNGSZENTRUM,ABT BIOPHYS MAKROMOL,NEUENHEIMER FELD 280 D-69120 HEIDELBERG GERMANY
Titolo Testata:
Journal of Molecular Biology
fascicolo: 5, volume: 278, anno: 1998,
pagine: 915 - 933
SICI:
0022-2836(1998)278:5<915:ASOTTA>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
NITROGEN REGULATOR-I; ESCHERICHIA-COLI; ATPASE ACTIVITY; DNA-BINDING; HYDRODYNAMIC PROPERTIES; RESPONSE REGULATOR; NUMERICAL-ANALYSIS; BOUNDARY ANALYSIS; RNA-POLYMERASE; ENHANCER;
Keywords:
ENHANCER; PROTEIN-DNA INTERACTION; TRANSCRIPTION; RNA POLYMERASE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
71
Recensione:
Indirizzi per estratti:
Citazione:
K. Rippe et al., "ASSOCIATION STATES OF THE TRANSCRIPTION ACTIVATOR PROTEIN NTRC FROM ESCHERICHIA-COLI DETERMINED BY ANALYTICAL ULTRACENTRIFUGATION", Journal of Molecular Biology, 278(5), 1998, pp. 915-933

Abstract

The transcription activator protein NtrC (nitrogen regulatory proteinC) can catalyze the transition of E. coli RNA polymerase complexed with the sigma(54) factor (RNAP.sigma(54)) from the closed complex (RNAP.sigma(54) bound at the promoter) to the open complex (melting of the promoter DNA). This process involves :phosphorylation of NtrC, assembly of a multimeric NtrC complex at the enhancer DNA sequence, interaction of this complex with promoter bound RNAP.sigma(54) via DNA looping,and hydrolysis of ATP. We have used analytical ultracentrifugation tostudy the different NtrC association states and to derive hydrodynamic models for the conformation of the various NtrC species. The following results were obtained. (i) The unphosphorylated wild-type protein formed a dimer with a measured molecular weight of 102(+/-3) kDa, whichcompares to a calculated molecular weight of 54 kDa for a monomer (concentration range studied 2 to 8 mu M NtrC monomer). (ii) in the unphosphorylated state one NtrC dimer was bound to one binding site as determined with DNA oligonucleotide duplexes containing one or two bindingsites (concentration range studied 50 to 1000 nM NtrC dimer). (iii) The data obtained at protein concentrations that were below the concentration of binding sites indicate that binding to the DNA duplex with two binding sites occurred with essentially no cooperativity. The experiments were conducted in the absence of ATP. (iv) The phosphorylated protein formed a specific complex at the DNA duplex with the enhancer sequence (two NtrC binding sites) that consisted of four dimers (concentration range studied 100 to 1000 nM NtrC dimer). (v) The formation ofthis octameric complex was highly cooperative, and the data suggest that two DNA strands could bind simultaneously to this complex. (vi) From the sedimentation data a model was derived in which the NtrC dimer adopts a V shaped structure with the DNA binding domains being locatedat the bottom and the two receiver domains at the top of the V. In this conformation higher order NtrC complexes can be stabilized by interaction between the phosphorylated receiver domain and the central activation domain of different NtrC dimers. (C) 1998 Academic Press Limited.

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Documento generato il 01/10/20 alle ore 03:56:29