Structural basis for -35 element recognition by σ4 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 (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 σ70σ470c and another from σSσ4Sc 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.