Conformation and DNA binding properties of a single-stranded DNA binding region of σ70 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 similar to 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 similar to 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.