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$\left({\sigma }_4^{70}c\right)$ and another from σ^S$\left({\sigma }_4^{S}c\right)$ 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 ${\sigma }_4^{S}c$ in complex with -35 element DNA revealed that ${\sigma }_4^{S}c$ adopts a similar structure as σ₄ 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), ${\sigma }_4^{70}c$ was demonstrated to recognize -35 element DNA similar to ${\sigma }_4^{S}c$ . Carr-Purcell-Meiboom-Gill relaxation dispersion analyses showed that the N-terminal helix and the β-flap-tip-helix of ${\sigma }_4^{70}c$ have a concurrent transient α-helical structure and DNA binding reduced the slow dynamics on ${\sigma }_4^{70}c$ . Finally, only ${\sigma }_4^{70}c$ 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.

Keywords: CPMG relaxation dispersion; NMR; PmrA response regulator; X-ray crystallography; domain 4 of σ factors.