Promoter recognition specificity of Corynebacterium glutamicum stress response sigma factors σD and σH deciphered using computer modeling and point mutagenesis

Abstract

This study aimed to reveal interactions of the stress response sigma subunits (factors) σ^D and σ^H of RNA polymerase and promoters in Gram-positive bacterium Corynebacterium glutamicum by combining wet-lab obtained data and in silico modeling. Computer modeling-guided point mutagenesis of C. glutamicum σ^H subunit led to the creation of a panel of σ^H variants. Their ability to initiate transcription from naturally occurring hybrid σ^D/σ^H-dependent promoter Pcg0441 and two control canonical promoters (σ^D-dependent PrsdA and σ^H-dependent PuvrD3) was measured and interpreted using molecular dynamics simulations of homology models of all complexes. The results led us to design the artificial hybrid promoter PD₃₅H₁₀ combining the −10 element of the PuvrD3 promoter and the −35 element of the PrsdA promoter. This artificial hybrid promoter PD_35-rsdAH_10-uvrD3 showed almost optimal properties needed for the bio-orthogonal transcription (not interfering with the native biological processes).