Structural basis for −35 element recognition by σ4 chimera proteins and their interactions with PmrA response regulator

© 2019 Wiley Periodicals, Inc. In class II transcription activation, the transcription factor normally binds to the promoter near the −35 position and contacts the domain 4 of σ factors (σ4) to activate transcription. However, σ4 of σ70 appears to be poorly folded on its own. Here, by fusing σ4 with the RNA polymerase β-flap-tip-helix, we constructed two σ4 chimera proteins, one from σ70 (σ704C) and another from σS (σS4C) of Klebsiella pneumoniae. The two chimera proteins well folded into a monomeric form with strong binding affinities for −35 element DNA. Determining the crystal structure of σS4C in complex with −35 element DNA revealed that σS4C adopts a similar structure as σ4 in the Escherichia coli RNA polymerase σS holoenzyme and recognizes −35 element DNA specifically by several conserved residues from the helix-turn-helix motif. By using nuclear magnetic resonance (NMR), σ704C was demonstrated to recognize −35 element DNA similar to σS4C. Carr-Purcell-Meiboom-Gill relaxation dispersion analyses showed that the N-terminal helix and the β-flap-tip-helix of σ704C have a concurrent transient α-helical structure and DNA binding reduced the slow dynamics on σ704C. Finally, only σ704C was shown to interact with the response regulator PmrA and its promoter DNA. The chimera proteins are capable of −35 element DNA recognition and can be used for study with transcription factors or other factors that interact with domain 4 of σ factors.