Conformational flexibility in sigma70 region 2 during transcription initiation.

Prokaryotic RNA polymerase holoenzyme is composed of core subunits (alpha(2)betabeta'omega) plus a sigma factor that confers promoter specificity allowing for regulation of gene expression. Holoenzyme is known to undergo several conformational changes during the multiple steps of transcription initiation. However, the effects of these changes on the functions of specific regions have not been well characterized. In this work, we addressed the role of possible conformational change in region 2 of Escherichia coli sigma(70) by engineering disulfide bonds that "lock" region 2.1 with region 2.2 and region 2.2 with region 2.3. When these mutant holoenzymes were characterized for gross defects in multiple-round transcription, we found that insertion of either disulfide bond did not result in a fundamental block, indicating that the disulfide-containing holoenzymes are active. However, both disulfide-containing holoenzymes exhibited defects in formation and stability of the open complex. Our results suggest that conformational flexibility within sigma(70) region 2 facilitates open complex formation and transcription initiation.