An A257V mutation in the bacillus subtilis response regulator Spo0A prevents regulated expression of promoters with low-consensus binding sites.

In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

Bacillus subtilis RNA polymerase recruits the transcription factor Spo0A approximately P to stabilize a closed complex during transcription initiation.

The Bacillus subtilis response regulator Spo0A approximately P activates transcription from the spoIIG promoter by stimulating a rate-limiting transition between the initial interaction of RNA polymerase with the promoter and initiation of RNA synthesis. Previous work showed that Spo0A exerts its effect on RNA polymerase prior to the formation of an open complex in which the DNA strands at the initiation site have been separated. To isolate the effect of Spo0A approximately P on events prior to DNA strand separation at spoIIG we studied RNA polymerase binding to DNA fragments that were truncated to contain only promoter sequences 5' to the -10 element by electrophoretic mobility shift assays. RNA polymerase bound to these fragments readily though highly reversibly, and polymerase-promoter complexes recruited Spo0A approximately P. Sequence-independent interactions between the RNA polymerase and the DNA upstream of the core promoter were important for RNA polymerase binding and essential for Spo0A approximately P recruitment, while sequence-specific Spo0A approximately P-DNA interactions positioned and stabilized RNA polymerase binding to the DNA. Spo0A approximately P decreased the dissociation rate of the complexes formed with truncated promoter templates which could contribute to the means by which Spo0A approximately P stimulates spoIIG expression.

Zebrafish Mnx proteins specify one motoneuron subtype and suppress acquisition of interneuron characteristics.

BACKGROUND: Precise matching between motoneuron subtypes and the muscles they innervate is a prerequisite for normal behavior. Motoneuron subtype identity is specified by the combination of transcription factors expressed by the cell during its differentiation. Here we investigate the roles of Mnx family transcription factors in specifying the subtypes of individually identified zebrafish primary motoneurons. RESULTS: Zebrafish has three Mnx family members. We show that each of them has a distinct and temporally dynamic expression pattern in each primary motoneuron subtype. We also show that two Mnx family members are expressed in identified VeLD interneurons derived from the same progenitor domain that generates primary motoneurons. Surprisingly, we found that Mnx proteins appear unnecessary for differentiation of VeLD interneurons or the CaP motoneuron subtype. Mnx proteins are, however, required for differentiation of the MiP motoneuron subtype. We previously showed that MiPs require two temporally-distinct phases of Islet1 expression for normal development. Here we show that in the absence of Mnx proteins, the later phase of Islet1 expression is initiated but not sustained, and MiPs become hybrids that co-express morphological and molecular features of motoneurons and V2a interneurons. Unexpectedly, these hybrid MiPs often extend CaP-like axons, and some MiPs appear to be entirely transformed to a CaP morphology. CONCLUSIONS: Our results suggest that Mnx proteins promote MiP subtype identity by suppressing both interneuron development and CaP axon pathfinding. This is, to our knowledge, the first report of transcription factors that act to distinguish CaP and MiP subtype identities. Our results also suggest that MiP motoneurons are more similar to V2 interneurons than are CaP motoneurons.

The Bacillus subtilis response regulator Spo0A stimulates sigmaA-dependent transcription prior to the major energetic barrier.

At the spoIIG promoter phosphorylated Spo0A (Spo0A approximately P) binds 0A boxes overlapping the -35 element, interacting with RNA polymerase to facilitate open complex formation. We have compared in vitro transcription from a series of heteroduplex templates containing denatured regions within the promoters. Transcription from heteroduplex templates with 12, 8, or 6 base pairs denatured was independent of Spo0A approximately P, but heteroduplexes with 4 or 2 base pairs denatured required Spo0A approximately P for maximal levels of transcription. Investigation of the thermal dependence of transcription suggested that strand separation was the primary thermodynamic barrier to transcription initiation but indicated that Spo0A approximately P does not reduce this energetic barrier. Kinetic assays revealed that Spo0A approximately P stimulated both the rate of formation of initiated complexes as well as increasing the number of complexes capable of initiating transcription. These results imply that Spo0A approximately P stimulates transcription at least in part by stabilizing the RNA polymerase-spoIIG complex until contacts between RNA polymerase and the -10 element induce strand separation.