IN-VITRO RECONSTITUTION OF TRANSCRIPTIONAL ANTITERMINATION BY THE SACT AND SACY PROTEINS OF BACILLUS-SUBTILIS

Expression of the sacPA and sacB genes of Bacillus subtilis is positiv ely modulated by transcriptional regulatory proteins encoded by the sa cT and sacY genes, respectively. Previous genetic studies led to the s uggestion that SacT and SacY function as nascent mRNA binding proteins preventing early termination of transcription at terminators located in the leader regions of the corresponding genes. Here we report the o verproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY. Using mRNA band migration r etardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of p urified SacT and SacY are demonstrated under in vitro conditions. The results establish for the first time that members of the BglG family o f antiterminators function in antitermination in the absence of other proteins in vitro. Purified SacT is shown to be phosphorylated by phos phoenolpyruvate in a phosphotransferase-catalyzed reaction dependent o n Enzyme I and HPr. Unexpectedly, the purified SacT is shown to be fun ctional in mRNA binding and in transcriptional antitermination indepen dently of its phosphorylation state.