In Escherichia coli, the AraC protein represses transcription from its own promoter, p C , and when associated with arabinose, activates transcription from three other promoters, p BAD , p E , and p FGH . Expression from all four of these promoters is also regulated by cyclic AMP-catabolite activator protein; however, the arrangement of the protein binding sites is not identical for each promoter. We are interested in determining how the AraC protein is able to activate p BAD , p E , and p FGH despite their differences. We have characterized the induction response of the wild-type arabinose operons from their native chromosomal locations by primer extension analysis. In this analysis, mRNA from the four arabinose operons plus an internal standard could all be assayed in the RNA obtained from a single sample of cells. We found that each of the operons shows a rapid, within 15 to 30 s, response to arabinose. We also found that the expression of araFGH is more sensitive to catabolite repression but not to arabinose concentration than are araE and araBAD. Finally, we have determined the relative levels of inducibility in wild-type cells of araBAD, araFGH, and araE to be 6.5, 5, and 1, respectively. These results provide a basis for subsequent studies to determine the mechanism(s) by which AraC protein activates transcription from the different arabinose promoters.Arabinose utilization in Escherichia coli requires the expression of the metabolic operon, araBAD (8,9,33), and expression of either the low-affinity transport operon, araE (22), or the high-affinity transport operon, araFGH (3,12,15). Induction of each of these operons normally requires the AraC protein (5) complexed with arabinose and the catabolite activator protein (CAP) complexed with cyclic AMP (cAMP). The regulatory protein binding sites and the transcription start sites at each of the arabinose-responsive promoters have been determined (10, 17, 18, 23, 31, 32). Studies of the araBAD promoter, p BAD , show that to activate transcription the AraC protein binding site must overlap the Ϫ35 region of the promoter by 4 bp (25) (Fig. 1). Further, the two half-sites recognized by the dimeric AraC protein must be in the same direct repeat orientation (4, 21) to activate transcription. These facts suggest that specific contacts are made between the AraC protein and RNA polymerase at p BAD . Providing further support for this theory is the almost identical arrangement of the protein binding sites for araBAD, araE, and araJ, a weak, arabinose-inducible promoter whose gene product is nonessential (24). Surprisingly, the araFGH promoter, p FGH , possesses a radically different structure (Fig. 1). In p FGH the CAP site, rather than the AraC site, overlaps the Ϫ35 recognition sequence of RNA polymerase. Additionally, the AraC sites in araFGH are arranged in the opposite direct repeat orientation (Fig. 1).This work is a first step in studying the mechanism(s) by which AraC regulates transcription at the araE promoter, p E , and at p FGH . In the present study, we ha...

The basic helix-loop-helix (bHLH) transcription factor family regulates numerous developmental events in eukaryotic cells. In the model system, C. elegans, thirty-seven bHLH proteins have been identified via genome-wide sequence analysis and fourteen have been genetically characterized to date. These proteins influence cell fate specification of neural lineages and differentiation of myogenic lineages and have distinct roles in somatic gonadogenesis. We report here on the molecular characterization of HLH-17, a protein whose putative bHLH domain is homologous to the mammalian bHLH proteins BETA3 and bHLHB5. The gene hlh-17 is transcriptionally active at all developmental stages, with the highest steady state accumulation of hlh-17 mRNA during embryogenesis. An upstream hlh-17 sequence drives expression of GFP in the sheath cells of the cephalic sensilla. Finally, animals that are defective in HLH-17 via RNAi display egg-laying defects, while those carrying null mutations in hlh-17 do not develop beyond the L2 stage and are less attracted to potassium and sodium ions. We propose that hlh-17 affects the ability of C. elegans to respond to food cues, with possible downstream effects on insulin-signaling genes involved in the normal development and reproductive viability of the worm.

In vertebrates and invertebrates, dopamine signaling modulates a wide variety of physical and behavioral functions and exerts these effects through heterotrimeric G proteins. The soil nematode Caenorhabditis elegans has been used to model dopamine signaling and reacts reproducibly to alterations in dopamine levels through eight well-characterized dopaminergic neurons located in the head. In C. elegans, the basic helix-loop-helix transcription factor HLH-17 is strongly and constitutively expressed in the glia cells that ensheath four of the dopaminergic neurons, yet it is not required for specification or development of either the glia or the neurons. In this study, we sought to determine whether HLH-17 functions in dopamine signaling. We found that, unlike wild-type animals, hlh-17 animals are resistant to the effects of exogenous dopamine on egg laying and mobility. hlh-17 animals are also defective in the basal slowing and gustatory plasticity behaviors that require functional dopamine signaling. We also found that the expression of the dopamine receptor genes dop-1, dop-2, and dop-3 and the RGS protein gene egl-10 is significantly reduced in hlh-17 animals. Together these results point to a role for HLH-17 in dopamine signaling in C. elegans.

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In Caenorhabditis elegans, the six proteins that make up the REF-1 family have been identified as functional homologs of the Hairy/Enhancer of Split (HES) proteins. These transcription factors act in both Notch dependent and Notch-independent pathways to regulate embryonic events during development; however, their post-embryonic functions are not well defined. As a first step toward understanding how the REF-1 family works together to coordinate post-embryonic events, we used gene expression microarray analysis to identify transcriptional targets of HLH-29 in L4/young adult stage animals. Here we show that HLH-29 targets are genes needed for the regulation of growth and lifespan, including genes required for oxidative stress response and fatty acid metabolism, and the ferritin genes, ftn-1 and ftn-2. We show that HLH-29 regulates ftn-1 expression via promoter sequences upstream of the iron-dependent element that is recognized by the hypoxia inducible factor, HIF-1. Additionally, hlh-29 mutants are more resistant to peroxide stress than wild-type animals and ftn-1(RNAi) animals, even in the presence of excess iron. Finally we show that HLH-29 acts parallel to DAF-16 but upstream of the microphthalmia transcription factor ortholog, HLH-30, to regulate ftn-1 expression under normal growth conditions.