Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein.
We have reconstituted Bacillus subtilis trp attenuation in vitro. Purification of the mtrB gene product (TRAP) to near homogeneity allowed us to demonstrate that addition of this protein plus L-tryptophan to template, RNA polymerase, and nucleoside triphosphates caused transcription termination in the trpEDCFBA leader region. TRAP acts by binding to the nascent transcript and preventing formation of an RNA antiterminator structure, thereby allowing terminator formation and transcription termination. Oligonucleotides complementary to segments of the antiterminator were used to demonstrate that formation of this RNA hairpin was responsible for transcription read-through. TRAP was found to be a 60-kDa multimeric protein composed of identical 6- to 8-kDa subunits, and its elution profile on a chromatographic column did not change in the presence of tryptophan.
Babitzke
P
P
Department of Biological Sciences, Stanford University, CA 94305.
Yanofsky
C
C
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D009876:Operon; D017433:Protein Structure, Secondary; D012329:RNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014364:Tryptophan / Q000494:pharmacology*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan; C079092:MtrB protein, Bacteria; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins