Characterization of a transitionally occupied state and thermal unfolding of domain 1.1 of s^A factor of RNA polymerase from Bacillus subtilis

sigma factors are essential parts of bacterial RNA polymerase (RNAP) as they allow to recognize promotor sequences and initiate transcription. Domain 1.1 of vegetative sigma factors occupies the primary channel of RNAP and also prevents binding of the sigma factor to promoter DNA alone. Here, we show that domain 1.1 of Bacillus subtilis sigma(A) exists in more structurally distinct variants in dynamic equilibrium. The major conformation at room temperature is represented by a previously reported well-folded structure solved by nuclear magnetic resonance (NMR), but 4% of the protein molecules are present in a less thermodynamically favorable state. We show that this population increases with temperature and we predict its significant elevation at higher but still biologically relevant temperatures. We characterized the minor state of the domain 1.1 using specialized methods of NMR. We found that, in contrast to the major state, the detected minor state is partially unfolded. Its propensity to form secondary structure elements is especially decreased for the first and third alpha helices, while the second alpha helix and ss strand close to the C-terminus are more stable. We also analyzed thermal unfolding of the domain 1.1 and performed functional experiments with full length sigma(A) and its shortened version lacking domain 1.1 ((sigma A_Delta 1:1)). The results revealed that while full length sA increases transcription activity of RNAP with increasing temperature, transcription with (sigma A_Delta 1:1) remains constant. In summary, this study reveals conformational dynamics of domain 1.1 and provides a basis for studies of its interaction with RNAP and effects on transcription regulation.