The TFE-induced transient native-like structure of the intrinsically disordered σ₄⁷⁰ domain of Escherichia coli RNA polymerase.

The transient folding of domain 4 of an E. coli RNA polymerase σ⁷⁰ subunit (rECσ₄⁷⁰) induced by an increasing concentration of 2,2,2-trifluoroethanol (TFE) in an aqueous solution was monitored by means of CD and heteronuclear NMR spectroscopy. NMR data, collected at a 30% TFE, allowed the estimation of the population of a locally folded rECσ₄⁷⁰ structure (CSI descriptors) and of local backbone dynamics ((15)N relaxation).

Backbone dynamics of TFE-induced native-like fold of region 4 of Escherichia coli RNA polymerase sigma70 subunit.

Folding of a recombinant protein rECsigma(70) (4) comprising domain 4 of E. coli RNA polymerase sigma(70) subunit, upon addition of 2,2,2-trifluoroethanol (TFE) to its aqueous solution, was monitored by heteronuclear NMR spectroscopy. The TFE-induced migration of resonance signals in a series of (15)N-HSQC spectra displayed sequence-dependent heterogeneity.

Equivalence of Mg2+ and Na+ ions in salt dependence of the equilibrium binding and dissociation rate constants of Escherichia coli RNA polymerase open complex.

Conflicting experimental data on the influence of Mg(2+) ions on the salt dependence of formation/dissociation of open transcription complex (RPo) of Escherichia coli RNA polymerase led us to carry systematic measurements of the dissociation rate constant (k(d)) and thermodynamic stability of complexes at lambdaP(R) and Pa promoters in a broad range of [NaCl] and [MgCl(2)] at 25, 31 and 37 degrees C, using fluorescence detected abortive transcription assay. Values of k(d) determined in MgCl(2) in the presence of heparin, as a commonly used anionic competitor, were shown to depend on heparin concentration whereas in NaCl this effect was not observed. Kinetics of dissociation was therefore determined in the course of salt-induced down-shift of the binding equilibrium.

Identification of activating region (AR) of Escherichia coli LysR-type transcription factor CysB and CysB contact site on RNA polymerase alpha subunit at the cysP promoter.

CysB is a LysR-type transcriptional regulator (LTTR) controlling the expression of numerous genes involved in bacterial sulphur assimilation via cysteine biosynthesis. Our previous mutational analysis of CysB identified several residues within the N-terminal domain crucial for DNA-binding function. Here, we focus on the functional significance of CysB residues localized in the turn between the alpha2 and alpha3 helices forming an N-terminal helix-turn-helix motif.

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit.

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.