Sporulation in Bacillus subtilis is governed by a cascade of alternative RNA polymerase sigma factors. We previously identified a small protein Fin that is produced under the control of the sporulation sigma factor σF to create a negative feedback loop that inhibits σF-directed gene transcription. Cells deleted for fin are defective for spore formation and exhibit increased levels of σF-directed gene transcription. Based on pull-down experiments, chemical crosslinking, bacterial two-hybrid experiments, and nuclear magnetic resonance chemical shift analysis, we now report that Fin binds to RNA polymerase and specifically to the coiled-coil region of the β’ subunit. The coiled-coil is a docking site for sigma factors on RNA polymerase, and evidence is presented that the binding of Fin and σF to RNA polymerase is mutually exclusive. We propose that Fin functions by a mechanism distinct from that of classic sigma factor antagonists (anti-σ factors), which bind directly to a target sigma factor to prevent its association with RNA polymerase, and instead functions to inhibit σF by competing for binding to the β’ coiled-coil.
Sigma () 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 = subunit. We have identified an amino acid substitution (L257P) in the coiled coil that markedly inhibits the function of H , the earliestacting 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 H -directed gene expression but not in the activity of the stress-response sigma factor B . Pulldown experiments showed that the mutant RNAP was defective in associating with H but could still associate with A and B . The differential effects of the L257P substitution on sigma factor binding to RNAP are likely due to a conformational change in the = coiled coil that is specifically detrimental for interaction with 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 factor. Bacteria contain a primary factor that directs transcription of housekeeping genes and alternative factors that direct transcription in response to environmental or developmental cues. We identified an amino acid substitution (L257P) in the B. subtilis = subunit whereby RNAP L257P associates with some factors ( A and B ) and enables vegetative cell growth but is defective in utilization of 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 P romoter recognition in bacteria is governed by the sigma () subunit of RNA polymerase (RNAP), which directly contacts Ϫ10 and Ϫ35 sequence elements upstream of the transcription start site. The primary or housekeeping sigma factor is called 70 in Escherichia coli and A in Bacillus subtilis. A wide variety of alternative bacterial and phage sigma factors can replace the housekeeping sigma factor to direct the recognition of alternative classes of promoters. Many of these alternative sigma
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