Promoter recognition specificity of Corynebacterium glutamicum stress response sigma factors σ D and σ H 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 P cg0441 and two control canonical promoters (σ D -dependent P rsdA and σ H -dependent P uvrD3 ) was measured and interpreted using molecular dynamics simulations of homology models of all complexes. The results led us to design the artificial hybrid promoter P D 35 H 10 combining the −10 element of the P uvrD3 promoter and the −35 element of the P rsdA promoter. This artificial hybrid promoter P D 35-rsdA H 10-uvrD3 showed almost optimal properties needed for the bio-orthogonal transcription (not interfering with the native biological processes).