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

• Published in: Journal of computer-aided molecular design Vol. 39; no. 1; p. 1
• Main Authors: Blumenstein, J., Dostálová, H., Rucká, L., Štěpánek, V., Busche, T., Kalinowski, J., Pátek, M., Barvík, I.
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.12.2025; Springer Nature B.V
• Subjects: Amino acids; Animal Anatomy; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological activity; Biological properties; Chemistry; Chemistry and Materials Science; Cloning; Computer Applications in Chemistry; Computer Simulation; Corynebacterium glutamicum - genetics; Design; DNA-Directed RNA Polymerases - chemistry; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; E coli; Enzymes; Gene Expression Regulation, Bacterial; Genes; Genomes; Histology; Homology; Modelling; Molecular dynamics; Molecular Dynamics Simulation; Morphology; Mutagenesis; Physical Chemistry; Plasmids; Point Mutation; Promoter Regions, Genetic; RNA polymerase; Sigma Factor - chemistry; Sigma Factor - genetics; Sigma Factor - metabolism; Stress, Physiological - genetics; Transcription, Genetic; Promoter; Bio-orthogonal transcription; Sigma factor; Corynebacterium
• ISSN: 0920-654X; 1573-4951; 1573-4951
• DOI: 10.1007/s10822-024-00577-x

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).