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

Abstract

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 σ470c and another from σS σ4Sc 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 σ4Sc in complex with −35 element DNA revealed that σ4Sc 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), σ470c was demonstrated to recognize −35 element DNA similar to σ4Sc . Carr‐Purcell‐Meiboom‐Gill relaxation dispersion analyses showed that the N‐terminal helix and the β‐flap‐tip‐helix of σ470c have a concurrent transient α‐helical structure and DNA binding reduced the slow dynamics on σ470c . Finally, only σ470c 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.