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Overexpression of the trp leader transcript (containing the trp TRAP binding site) in trans has been shown to titrate TRAP and lead to increased trp gene expression (). If the site identified overlapping the beginning of the ycbK gene is able to bind TRAP, overexpression of this region should yield a transcript segment that would sequester TRAP and increase trp operon expression. However, at the high tryptophan levels needed to activate TRAP, transcription normally would cease at the terminator preceding yczA, therefore the putative TRAP binding segment of the transcript would not be synthesized. To overcome this barrier, a portion of the leader region terminator structure was deleted by site-directed mutagenesis and the construct was introduced into the homologous chromosomal locus. In this construct (ΔTerm-592) transcription should continue into the structural gene regions, ending at nucleotide 592, regardless of the level of tryptophan present. Expression of a trpE-lacZ fusion integrated into the amyE locus of the same strain was monitored and found to be significantly elevated (Table ), although not to the same degree as observed in an mtrB mutant strain. This finding would indicate that transcription of the yczA-ycbK operon in the presence of high levels of tryptophan does yield a transcript that is capable of binding TRAP, thus making it unavailable to fully down-regulate trpE expression. Introduction of the terminator-deletion construct into an mtrB strain did not result in higher trpE expression, indicating that the effect is mediated through TRAP.

Oct 27, 2008 · Describe the operon hypothesis (Ch

In Escherichia coli and many other bacterial species, transcription of the genes responsible for tryptophan formation is regulated by repression and transcription attenuation in response to changes in the concentration of tryptophan and charged tRNATrp, respectively (). Because these bacterial species have regulatory mechanisms that sense both tryptophan and charged tRNATrp, it seemed likely that B. subtilis also would sense these two intermediates in protein synthesis. The transcription attenuation mechanism that regulates transcription of the trp operon of B. subtilis relies on tryptophan as its signal. However, it was observed previously in studies with a temperature-sensitive tryptophanyl-tRNA synthetase mutant (), and confirmed recently (), that the extent of charging of tRNATrp also markedly influences trp operon expression. It also was shown previously that this second tRNATrp-sensing mechanism operates through the TRAP system (). It was postulated that increased trp operon transcription could be the result of either uncharged tRNATrp inhibition of TRAP function or increased synthesis of some transcript capable of binding and sequestering TRAP, reducing its availability (). In this study we searched for the participants in this hypothetical tRNATrp-mediated regulatory mechanism. We identified an operon that is regulated by tRNATrp that specifies a transcript with a potential TRAP binding site. The operon contains genes of unknown function, yczA and ycbK.

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Strains of Bacillus subtilis containing a temperature-sensitive tryptophanyl-tRNA synthetase produce elevated levels of the tryptophan pathway enzymes, when grown at high temperatures in the presence of excess tryptophan. This increase is because of reduced availability of the tryptophan-activated trp RNA-binding attenuation protein (TRAP). To test the hypothesis that this elevated trp gene expression was caused by the overproduction of a transcript capable of binding and sequestering TRAP, a computer program was designed to search the B. subtilis genome sequence for additional potential TRAP binding sites. A region containing a stretch of (G/A)AG trinucleotide repeats, characteristic of a TRAP binding site, was identified in the yczA-ycbK operon. We show that transcriptional regulation of the yczA-ycbK operon is controlled by the T-box antitermination mechanism in response to the level of uncharged tRNATrp, and that the presence of a trpS1 mutant allele increases production of the yczA-ycbK transcript. Elevated yczA-ycbK expression was shown to activate transcription of the trp operon. Deletion of the yczA-ycbK operon abolishes the trpS1 effect on trp gene expression. The purpose of increasing expression of the genes of tryptophan biosynthesis in the trpS mutant would be to provide additional tryptophan to overcome the charged tRNATrp deficiency. Therefore, in B. subtilis, as in Escherichia coli, transcription of the tryptophan biosynthetic genes is regulated in response to changes in the extent of charging of tRNATrp as well as the availability of tryptophan.