An Amino Acid Substitution in RNA Polymerase That Inhibits the Utilization of an Alternative Sigma Factor

Abstract

Sigma (σ) factors direct gene transcription by binding to and determining the promoter recognition specificity of RNA polymerase (RNAP) in bacteria. Genes transcribed under the control of alternative sigma factors allow cells to respond to stress and undergo developmental processes, such as sporulation in Bacillus subtilis, in which gene expression is controlled by a cascade of alternative sigma factors. Binding of sigma factors to RNA polymerase depends on the coiled-coil (or clamp helices) motif of the β' subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of σ^H, the earliest-acting alternative sigma factor in the sporulation cascade. Cells with this mutant RNAP exhibited an early and severe block in sporulation but not in growth. The mutant was strongly impaired in σ^H-directed gene expression but not in the activity of the stress-response sigma factor σ^B Pulldown experiments showed that the mutant RNAP was defective in associating with σ^H but could still associate with σ^A and σ^B The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the β' coiled coil that is specifically detrimental for interaction with σ^H This is the first example, to our knowledge, of an amino acid substitution in RNAP that exhibits a strong differential effect on a particular alternative sigma factor.IMPORTANCE In bacteria, all transcription is mediated by a single multisubunit RNA polymerase (RNAP) enzyme. However, promoter-specific transcription initiation necessitates that RNAP associates with a σ factor. Bacteria contain a primary σ factor that directs transcription of housekeeping genes and alternative σ factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis β' subunit whereby RNAP^L257P associates with some σ factors (σ^A and σ^B) and enables vegetative cell growth but is defective in utilization of σ^H and is consequently blocked for sporulation. To our knowledge, this is the first identification of an amino acid substitution within the core enzyme that affects utilization of a specific sigma factor.

Keywords: RNA polymerase; sigma factor; sporulation; transcription.