Reconstitution of Bacillus subtilis trp attenuation in vitro with TRAP, the trp RNA-binding attenuation protein

Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):133-7. doi: 10.1073/pnas.90.1.133.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacillus subtilis / genetics*
  • Bacillus subtilis / metabolism
  • Bacterial Proteins*
  • Base Sequence
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Gene Expression Regulation, Bacterial
  • Genes, Bacterial
  • Macromolecular Substances
  • Molecular Sequence Data
  • Molecular Weight
  • Nucleic Acid Conformation
  • Operon*
  • Protein Structure, Secondary
  • 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*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Repressor Proteins / genetics
  • Sequence Homology, Amino Acid
  • Templates, Genetic
  • Terminator Regions, Genetic*
  • Transcription Factors / genetics
  • Transcription Factors / isolation & purification
  • Transcription Factors / metabolism*
  • Transcription, Genetic* / drug effects
  • Tryptophan / pharmacology*

Substances

  • Bacterial Proteins
  • Macromolecular Substances
  • MtrB protein, Bacteria
  • RNA, Bacterial
  • RNA, Messenger
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Repressor Proteins
  • TRPR protein, E coli
  • Transcription Factors
  • Tryptophan