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 antiterminator 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 6362-6370 |
| Number of pages | 9 |
| Journal | Journal of bacteriology |
| Volume | 177 |
| Issue number | 22 |
| DOIs | |
| State | Published - Jan 1 1995 |
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All Science Journal Classification (ASJC) codes
- Microbiology
- Molecular Biology
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trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon. / Merino, E.; Babitzke, P.; Yanofsky, C.
In: Journal of bacteriology, Vol. 177, No. 22, 01.01.1995, p. 6362-6370.Research output: Contribution to journal › Article
TY - JOUR
T1 - trp RNA-binding attenuation protein (TRAP)-trp leader RNA interactions mediate translational as well as transcriptional regulation of the Bacillus subtilis trp operon
AU - Merino, E.
AU - Babitzke, P.
AU - Yanofsky, C.
PY - 1995/1/1
Y1 - 1995/1/1
N2 - 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 antiterminator 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.
AB - 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 antiterminator 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.
UR - http://www.scopus.com/inward/record.url?scp=0028871292&partnerID=8YFLogxK
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U2 - 10.1128/jb.177.22.6362-6370.1995
DO - 10.1128/jb.177.22.6362-6370.1995
M3 - Article
C2 - 7592410
AN - SCOPUS:0028871292
VL - 177
SP - 6362
EP - 6370
JO - Journal of Bacteriology
JF - Journal of Bacteriology
SN - 0021-9193
IS - 22
ER -