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 positively modulated by transcriptional regulatory proteins encoded by the sacT and sacY genes, respectively. Previous genetic studies led to the suggestion 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 overproduction, purification to near homogeneity, and characterization of the two antiterminators, SacT and SacY. Using mRNA band migration retardation assays and a reconstituted transcriptional antitermination system, the mRNA binding functions and antitermination activities of purified SacT and SacY are demonstrated under in vitro conditions. The results establish for the first time that members of the BglG family of antiterminators function in antitermination in the absence of other proteins in vitro. Purified SacT is shown to be phosphorylated by phosphoenolpyruvate in a phosphotransferase-catalyzed reaction dependent on Enzyme I and HPr. Unexpectedly, the purified SacT is shown to be functional in mRNA binding and in transcriptional antitermination independently of its phosphorylation state.
Arnaud
M
M
Unité de Biochimie Microbienne, Institut Pasteur, URA 1300 Centre National de la Recherche Scientifique 25, rue du Docteur Roux-75724 Paris Cedex 15, France.
Débarbouillé
M
M
Rapoport
G
G
Saier
M H
MH
Reizer
J
J
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C076894:SacY protein, Bacillus subtilis; D014157:Transcription Factors