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) sigma(D) and sigma(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 sigma(H) subunit led to the creation of a panel of sigma(H) variants. Their ability to initiate transcription from naturally occurring hybrid sigma(D)/sigma(H)-dependent promoter Pcg0441 and two control canonical promoters (sigma(D)-dependent PrsdA and sigma(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 PD(35)H(10) combining the -10 element of the PuvrD3 promoter and the -35 element of the PrsdA promoter. This artificial hybrid promoter PD(35-rsdA)H(10-uvrD3) showed almost optimal properties needed for the bio-orthogonal transcription (not interfering with the native biological processes).