PMID:7592410

Citation	Merino, E, Babitzke, P and Yanofsky, C (1995) trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon. J. Bacteriol. 177:6362-70
Abstract	Expression of the Bacillus subtilis trpEDCFBA operon has been shown to be regulated by transcription attenuation in response to the availability of L-tryptophan. Regulation is mediated by the tryptophan-activated trp RNA-binding attenuation protein, TRAP, the product of mtrB. Formation of mutually exclusive RNA anti-terminator and terminator structures within trp leader RNA determines whether transcription will terminate in the leader region of the operon. Previous studies suggested that transcripts that escape termination are subject to translational regulation via the formation of a secondary structure that blocks ribosome access to the trpE ribosome-binding site. To assess the relative importance of these postulated events in trp operon regulation, we used site-directed mutagenesis to alter the putative elements involved in transcriptional and translational control. Using a trpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB+ and mtrB strains, during growth in the presence and absence of excess tryptophan. To verify that the altered regulatory regions behaved as expected, we carried out in vitro transcription assays with the wild-type and altered leader region templates and performed oligonucleotide competition assays with an oligonucleotide complementary to a segment of the transcription terminator. Our results establish that binding of TRAP to leader RNA regulates of transcription termination in the trp operon over about an 88-fold range and regulates translation of trpE over about a 13-fold range. The roles played by different trp leader RNA segments in mediating transcriptional and translational regulation are documented by our findings.
Links	PubMed PMC177485
Keywords	Bacillus subtilis/genetics; Bacterial Proteins; Base Sequence; Gene Expression Regulation, Bacterial/genetics; Molecular Sequence Data; Mutation; Nucleic Acid Conformation; Operon/genetics; Protein Biosynthesis/genetics; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Messenger/chemistry; RNA, Messenger/genetics; RNA, Messenger/metabolism; RNA-Binding Proteins/metabolism; Recombinant Fusion Proteins/biosynthesis; Terminator Regions, Genetic/genetics; Transcription Factors/metabolism; Transcription, Genetic/genetics; Tryptophan/biosynthesis
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BACSU:MTRB		GO:0031555: transcriptional attenuation	ECO:0000314:		P		Figure 4, in vitro experiment using a complementary oligo to the DNA terminator hairpin in a transcription assay, showing a decrease in % termination, which supports the developed model of attenuation done by TRAP	complete CACAO 11088
BACSU:MTRB		GO:0060566: positive regulation of DNA-templated transcription, termination	ECO:0000314:		P		Figure 3. In vitro transcription assay, in the presence of TRAP and L-tryptophan transcription termination greatly increases.	complete CACAO 11089

References

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