Reconstitution of Bacillus subtilis trp Attenuation in vitro with TRAP, the trp RNA-Binding Attenuation Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 90; no. 1; pp. 133 - 137
• Main Authors: Babitzke, Paul, Yanofsky, Charles
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.01.1993; National Acad Sciences; National Academy of Sciences
• Subjects: Amino Acid Sequence; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacteria; Bacterial Proteins; Base Sequence; Binding sites; Biological and medical sciences; Cloning, Molecular; DNA; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gels; Gene Expression Regulation, Bacterial; Genes; Genes, Bacterial; Macromolecular Substances; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Molecular Weight; Nucleic Acid Conformation; Nucleotides; Oligonucleotides; Operon; Operons; Protein Structure, Secondary; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Repressor Proteins - genetics; Ribonucleic acid; RNA; RNA, Bacterial - biosynthesis; RNA, Bacterial - genetics; RNA, Messenger - chemistry; RNA, Messenger - genetics; RNA-Binding Proteins - genetics; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Sequence Homology, Amino Acid; Templates, Genetic; Terminator regions; Terminator Regions, Genetic; Transcription Factors - genetics; Transcription Factors - isolation & purification; Transcription Factors - metabolism; Transcription, Genetic - drug effects; Transcription. Transcription factor. Splicing. Rna processing; Transcriptional regulatory elements; Tryptophan - pharmacology; In vitro transcription; Purification; Leader sequence; Bacillus subtilis; Operon; RNA binding protein; Tryptophan; Bacillaceae; Transcription termination; Regulation(control); Bacillales; Transcription attenuation; Reconstituted system; Bacteria; Trans acting regulatory factor
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.1.133

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.