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In vitro experiments have shown that the genes of the de novo pyrimidine biosynthetic pathway of Bacillus subtilis, the pyr genes, are regulated by a transcriptional attenuation mechanism. Specific regulatory sequences (binding loops, BLs) are located within three untranslated leader sequences at the beginning of pyr mRNA. These binding loops, BL1, BL2 and BL3, act as anti-antiterminators of transcription when stabilized by the regulator protein PyrR. In this work, the interaction of PyrR with BL1, BL2 and BL3 was qualitatively and quantitatively analysed in vivo using the yeast three-hybrid system. The results indicate that PyrR specifically binds to BL1, BL2 and BL3. Furthermore, the data suggest that the strength of interaction between PyrR and the three different BLs in vivo is within the same dimension. The yeast three-hybrid system also proved to be useful for the rapid analysis of structural requirements for PyrR-BL binding. Point mutations within the predicted critical regions of BL1, BL2 and BL3 led to drastically reduced binding of PyrR. In summary, it is shown that the yeast three-hybrid system is well suited to qualitatively and quantitatively analyse bacterial regulatory systems that are based on factor-independent transcriptional attenuation.

RibR is a minor cryptic flavokinase (EC 2.7.1.26) of the Gram-positive bacterium Bacillus subtilis with an unknown cellular function. The flavokinase activity appears to be localized to the N-terminal domain of the protein. Using the yeast three-hybrid system, it was shown that RibR specifically interacts in vivo with the nontranslated wild-type leader of the mRNA of the riboflavin biosynthetic operon. This interaction is lost partially when a leader containing known cis-acting deregulatory mutations in the so-called RFN element is tested. The RFN element is a sequence within the rib-leader mRNA reported to serve as a receptor for an FMN-dependent ‘riboswitch’. In RibR itself, interaction was localized to the carboxy-terminate part of the protein, a segment of unknown function that does not show similarity to other proteins in the public databases. Analysis of a ribR-defective strain revealed a mild deregulation with respect to flavin (riboflavin, FMN and FAD) biosynthesis. The results indicate that the RNA-binding protein RibR may be involved in the regulation of the rib genes. [ABSTRACT FROM AUTHOR]

The riboflavin analog roseoflavin is an antibiotic produced by Streptomyces davawensis. Riboflavin transporters are responsible for roseoflavin uptake by target cells. Roseoflavin is converted to the flavin mononucleotide (FMN) analog roseoflavin mononucleotide (RoFMN) by flavokinase and to the flavin adenine dinucleotide (FAD) analog roseoflavin adenine dinucleotide (RoFAD by FAD synthetase. In order to study the effect of RoFMN and RoFAD in the cytoplasm of target cells, Escherichia coli was used as a model. E. coli is predicted to contain 38 different FMN- or FAD-dependent proteins (flavoproteins). These proteins were overproduced in recombinant E. coli strains grown in the presence of sublethal amounts of roseoflavin. The flavoproteins were purified and analyzed with regard to their cofactor contents. It was found that 37 out of 38 flavoproteins contained either RoFMN or RoFAD. These cofactors have different physicochemical properties than FMN and FAD and were reported to reduce or completely abolish flavoprotein function. [ABSTRACT FROM AUTHOR]