Core RNA polymerase from E. coli induces a major change in the domain arrangement of the sigma 70 subunit.

Luminescence resonance energy transfer measurements were used to show that binding of E. coli core RNA polymerase induced major changes in interdomain distances in the sigma 70 subunit. The simplest model describing core-induced changes in sigma 70 involves a movement of the conserved region 1 by approximately 20 A and the conserved region 4.2 by approximately 15 A with respect to conserved region 2. The core-induced movement of region 1 (autoinhibition domain) and region 4.2 (DNA-binding domain) provides structural rationale for allosteric regulation of sigma 70 DNA binding properties by the core and suggests that this regulation may not only involve directly the autoinhibition domain of sigma 70 but also could involve a modulation of spacing between DNA-binding domains of sigma 70 induced by binding of core RNAP.

Conformational changes of Escherichia coli RNA polymerase sigma70 factor induced by binding to the core enzyme.

Mutants of RNA polymerase sigma70 subunit from Escherichia coli with unique cysteine residues engineered into conserved region 1 (autoinhibition domain of sigma70), region 2.4 (-10 DNA element binding domain), region 4.2 (-35 DNA element binding domain), and a nonconserved region between regions 1 and 2 were prepared. The chemical reactivity of the cysteine at each position was determined for free sigma70 and sigma70 in complex with the core polymerase and was used as a measure of a conformational response of a particular region of the protein to an interaction with the core polymerase. Both increases and decreases in cysteine reactivity were observed in the presence of core polymerase at several positions in sigma70, providing direct physical evidence for modulation of sigma70 conformation by the core enzyme. Binding of the core polymerase resulted in increased solvent exposure of DNA binding domains of sigma70 and in more complex changes in the autoinhibition domain (region 1). Similar conformational changes in sigma70 were detected using fluorescence probes covalently attached to cysteine residues engineered into sigma70. Thus, the results obtained provided physical evidence supporting a model in which core enzyme allosterically regulates DNA binding activity of sigma70 by "unmasking" its DNA binding domains.

Conformation and DNA binding properties of a single-stranded DNA binding region of sigma 70 subunit from Escherichia coli RNA polymerase are modulated by an interaction with the core enzyme.

A derivative of the sigma 70 subunit from Escherichia coli RNA polymerase with specific fluorescence probes in conserved region 2.3 (DNA "melting motif") was prepared by replacing tryptophan residues at positions 314 and 326 of the wild-type sigma 70 with alanine. The remaining two tryptophan residues (Trp 433 and 434) of [Ala 314, 326]sigma 70 were biosynthetically replaced with 5-hydroxy-tryptophan (5OHTrp), a fluorescent tryptophan analogue with unique emission that can be selectively observed both in free 5OHTrp[Ala 314, 326]sigma 70 as well as in 5OHTrp[Ala 314, 326]sigma 70 bound to the core RNA polymerase. Fluorescence quenching experiments revealed that positions 433 and 434 were solvent exposed in free 5OHTrp[Ala314, 326]sigma 70. The binding of sigma 70 to core polymerase reduced the solvent exposure of these residues. In the presence of single-stranded oligonucleotides, fluorescence of 5OHTrp at position 433 and 434 was quenched approximately 65% and these residues became inaccessible to the solvent. Using fluorescence of 5OHTrp at positions 433 and 434 as a specific signal of DNA binding, we show that free sigma 70 bound single-stranded DNA weakly and did not discriminate between nontemplate and template strand of promoter DNA. Binding of sigma 70 to the core increased the affinity for binding nontemplate DNA, whereas the affinity to template or "nonspecific" DNA was reduced, resulting in a holoenzyme which could bind nontemplate strand approximately 200-fold better then the template strand. We concluded that Trp 433 and 434 of sigma 70 are located within a single-stranded DNA binding region of sigma 70 and that binding of sigma 70 to the core enzyme induced conformational changes in a single-stranded DNA binding region of the protein. As a consequence of these conformational changes, sigma 70 subunit gains the specificity for the nontemplate strand of the melted region in the "open" complex.