Structural basis for −35 element recognition by σ 4 chimera proteins and their interactions with PmrA response regulator

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 (σ 4 ) to activate transcription. However, σ 4 of σ 70 appears to be poorly folded on its own. Here, by fusing σ 4 with the RNA polymerase β‐flap‐tip‐helix, we constructed two σ 4 chimera proteins, one from σ 70σ 4 70 cand another from σ Sσ 4 S cof 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 σ 4 S c in complex with −35 element DNA revealed that σ 4 S c adopts a similar structure as σ 4 in the Escherichia coli RNA polymerase σ S holoenzyme and recognizes −35 element DNA specifically by several conserved residues from the helix‐turn‐helix motif. By using nuclear magnetic resonance (NMR), σ 4 70 c was demonstrated to recognize −35 element DNA similar to σ 4 S c . Carr‐Purcell‐Meiboom‐Gill relaxation dispersion analyses showed that the N‐terminal helix and the β‐flap‐tip‐helix of σ 4 70 c have a concurrent transient α‐helical structure and DNA binding reduced the slow dynamics on σ 4 70 c . Finally, only σ 4 70 c 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.