Architecture of a complex between the σ70 subunit of Escherichia coli RNA polymerase and the nontemplate strand oligonucleotide. Luminescence resonance energy transfer study

Abstract

We used luminescence energy transfer measurements to determine the localization of 5'- and 3'-ends of a 12-nucleotide nontemplate strand oligonucleotide bound to σ70 holoenzyme. Five single reactive cysteine mutants of σ70 (cysteine residues at positions 1, 59, 366, 442, and 596) were labeled with a europium chelate fluorochrome (donor). The oligonucleotide was modified at the 5'- or at the 3'-end with Cy5 fluorochrome (acceptor). The energy transfer was observed upon complex formation between the donor-labeled σ70 holoenzyme and the acceptor- labeled nontemplate strand oligonucleotide, whereas no interaction was observed with the template strand oligonucleotide. The oligonucleotide was bound in one preferred orientation. This observation together with the sequence specificity of single-stranded oligonucleotide interaction suggests that two mechanisms of discrimination between the template and nontemplate strand are used by σ70: sequence specificity and strand polarity specificity. The bound oligonucleotide was found to be close to residue 442, confirming that the single-stranded DNA binding site of σ70 is located in an α-helix containing residue 442. The 5'-end of the oligonucleotide was oriented toward the COOH terminus of the helix.