Architecture of a complex between the sigma(70) subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide - Luminescence resonance energy transfer study

We used luminescence energy transfer measurements to determine the localiza tion of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleoti de bound to sigma(70) holoenzyme, Five single reactive cysteine mutants of sigma(70) (cysteine residues at positions 1, 59, 366, 442, and 596) were la beled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the S'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labe led sigma(70) holoenzyme and the acceptor-labeled nontemplate strand oligon ucleotide, whereas no interaction was observed with the template strand oli gonucleotide. The oligonucleotide was bound in one preferred orientation. T his observation together with the sequence specificity of single-stranded o ligonucleotide interaction suggests that two mechanisms of discrimination b etween the template and nontemplate strand are used by sigma(70): sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA b inding site of sigma(70) is located in an cu-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.