The Bacillus subtilis genes for ribonucleotide reductase are similar to the genes for the second class I NrdE/NrdF enzymes of Enterobacteriaceae

We have cloned and sequenced the nrd (nucleotide reductase) locus of Bacillus subtilis. The locus seems to be organized in an operon comprising four ORFs. The first three encode polypeptides highly similar to the product of the coding sequences characterizing the nrdEF operons of Enterobacteriaceae. The sequencing of the conditional lethal mutation ts-A13, localized in the nrdE cistron, and the lethality of insertional mutations targeted in the internal region of nrdE and nrdF, demonstrated the essential role of this locus. The fourth ORF, ymaB, part of the putative operon, which is not similar to any known protein, is also essential. The regulation of expression of the operon, monitored by lacZ transcriptional fusions, is similar to the regulation of the functionally relevant nrdAB operon of Escherichia coli. The operon was induced by thymidine starvation and its expression was directly or indirectly affected by RecA function. Genetic and functional analysis strongly indicates that in B. subtilis the class I ribonucleotide reductase encoded by this nrd operon is evolutionarily distant from the homologous class I enzyme of Enterobacteria

14-3-3 proteins regulate the potassium channel KAT1 by dual modes

KAT1 is a cloned plant potassium channel belonging to the superfamily of Shaker-like Kv channels. Previous studies have shown that 14-3-3 proteins significantly increase KAT1 current by modifying the channel open probability. Employing a 14-3-3 scavenger construct to lower the long-term availability of endogenous 14-3-3 proteins, we found that 14-3-3 proteins not only control the voltage dependency of the channel but also the number of channels in the plasma membrane

Bacillus licheniformis Anti-TRAP can assemble into two types of dodecameric particles with the same symmetry but inverted orientation of trimers

Anti-TRAP (AT) protein regulates expression of tryptophan biosynthetic genes by binding to the trp RNA-binding attenuation protein (TRAP) and preventing its interaction with RNA. Bacillus subtilis AT forms trimers that can either interact with TRAP or can further assemble into dodecameric particles. To determine which oligomeric forms are preserved in AT proteins of other Bacilli we studied Bacillus licheniformis AT which shares 66% sequence identity with the B. subtilis protein. We show that in solution B. licheniformis AT forms stable trimers. In crystals, depending on pH, such trimers assemble into two different types of dodecameric particles, both having 23 point group symmetry. The dodecamer formed at pH 6.0 has the same conformation as previously observed for B. subtilis AT. This dodecamer contains a large internal chamber with the volume of ∼700 Å3, which is lined by the side chains of twelve valine residues. The presence of the hydrophobic chamber hints at the possibility that the dodecamer formation could be induced by binding of a ligand. Interestingly, in the dodecamer formed at pH 8.0 all trimers are turned inside out relatively to the form observed at pH 6.0

NusA-stimulated RNA polymerase pausing and termination participates in the Bacillus subtilis trp operon attenuation mechanism in vitro

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translation control mechanisms. Both mechanisms require the binding of tryptophan-activated TRAP to the 11 (G/U)AG-repeat segment in the trp leader transcript. To promote termination, TRAP must bind to the nascent RNA before the antiterminator structure forms. Because only 20 nucleotides separate the TRAP-binding site from the 3′ end of the antiterminator, TRAP has a short time frame to control this regulatory decision. Synchronization of factor binding and/or RNA folding with the RNA polymerase position is a major challenge in all attenuation mechanisms. Because RNA polymerase pausing allows this synchronization in many attenuation mechanisms, we performed experiments in vitro to determine whether pausing participates in the B. subtilis trp attenuation mechanism. We identified two NusA-stimulated pause sites in the trp leader region. Formation of pause hairpins participates in pausing at both positions. The first pause occurred at the nucleotide just preceding the critical overlap between the alternative antiterminator and terminator structures. TRAP binding to transcripts containing preexisting pause complexes releases RNA polymerase, suggesting that pausing provides additional time for TRAP to bind and promote termination. The second pause is downstream from the trp leader termination point, raising the possibility that this pause event participates in the trpE translation control mechanism. NusA also increases the efficiency of termination in the trp leader region and shifts termination one nucleotide upstream. Finally, NusA-stimulated termination is cooperative, suggesting that binding of multiple NusA molecules influences termination