The Prosequence of Pro-ς K Promotes Membrane Association and Inhibits RNA Polymerase Core Binding

Abstract

ABSTRACT Pro-ς K is the inactive precursor of ς K , a mother cell-specific sigma factor responsible for the transcription of late sporulation genes of Bacillus subtilis . Upon subcellular fractionation, the majority of the pro-ς K was present in the membrane fraction. The rest of the pro-ς K was in a large complex that did not contain RNA polymerase core subunits. In contrast, the majority of the ς K was associated with core RNA polymerase. Virtually identical fractionation properties were observed when pro-ς E was analyzed. Pro-ς K was completely solubilized from the membrane fraction and the large complex by Triton X-100 and was partially solubilized from the membrane fraction by NaCl and KSCN. The membrane association of pro-ς K did not require spoIVF gene products, which appear to be located in the mother cell membrane that surrounds the forespore, and govern pro-ς K processing in the mother cell. Furthermore, pro-ς K associated with the membrane when overproduced in vegetative cells. Overproduction of pro-ς K in sporulating cells resulted in more pro-ς K in the membrane fraction. In agreement with the results of cell fractionation experiments, immunofluorescence microscopy showed that pro-ς K was localized to the mother cell membranes that surround the mother cell and the forespore in sporulating wild-type cells and mutant cells that do not process pro-ς K . Treatment of extracts with 0.6 M KCl appeared to free most of the pro-ς K and ς K from other cell constituents. After salt removal, ς K , but not pro-ς K , reassociated with exogenous core RNA polymerase to form holoenzyme. These results suggest that the prosequence inhibits RNA polymerase core binding and targets pro-ς K to the membrane, where it may interact with the processing machinery.