AdpA is a key regulator of morphological differentiation in Streptomyces. In contrast to Streptomyces griseus, relatively little is known about AdpA protein functions in Streptomyces coelicolor. Here, we report for the first time the translation accumulation profile of the S. coelicolor adpA (adpA Sc ) gene; the level of S. coelicolor AdpA (AdpA Sc ) increased, reaching a maximum in the early stage of aerial mycelium formation (after 36 h), and remained relatively stable for the next several hours (48 to 60 h), and then the signal intensity decreased considerably. AdpA Sc specifically binds the adpA Sc promoter region in vitro and in vivo, suggesting that its expression is autoregulated; surprisingly, in contrast to S. griseus, the protein presumably acts as a transcriptional activator. We also demonstrate a direct influence of AdpA Sc on the expression of several genes whose products play key roles in the differentiation of S. coelicolor: STI, a protease inhibitor; RamR, an atypical response regulator that itself activates expression of the genes for a small modified peptide that is required for aerial growth; and ClpP1, an ATP-dependent protease. The diverse influence of AdpA Sc protein on the expression of the analyzed genes presumably results mainly from different affinities of AdpA Sc protein to individual promoters.Streptomycetes, GC-rich Gram-positive soil bacteria known for their ability to produce many valuable antibiotics and other secondary metabolites, undergo complex morphological differentiation (4, 10). The genome of the model species Streptomyces coelicolor A3(2) was the first among the Streptomycetes to be completely sequenced (2).Streptomyces bacteria grow by tip extension and hyphal branching to form a dense mycelial network of vegetative hyphae. In response to nutrient depletion and other signals, the vegetative mycelium is partially self-cannibalized by a nuclease(s) and protease(s) to supply nutrients for the growth of aerial hyphae, which subsequently transform into long chains of spores (3,27). This morphological differentiation, which is usually accompanied by the production of secondary metabolite(s), is controlled by multilevel regulatory mechanisms. A key coordinating role in the regulation of morphological differentiation is played by the protein AdpA, which was originally discovered in Streptomyces griseus (37,38). In all Streptomyces genomes sequenced so far, translation of adpA mRNA depends on a leucyl-tRNA for a rarely used TTA codon; the tRNA UAA Leu is encoded by the bldA gene required for aerial mycelium formation. AdpA belongs to the AraC/XylS family of transcription regulators, whose members contain a dual helix-turn-helix (HTH) motif in the C-terminal DNA binding domain. In S. griseus, AdpA activates a number of genes whose products are required for morphological development and for secondary metabolites synthesis (e.g., streptomycin) (12, 28). During vegetative growth, the transcription of S. griseus adpA (adpA Sg ) is repressed by ArpA, the receptor protein for the signal...

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, presumably utilizes fatty acids as a major carbon source during infection within the host. Metabolism of even-chain-length fatty acids yields acetyl-CoA, whereas metabolism of odd-chain-length fatty acids additionally yields propionyl-CoA. Utilization of these compounds by tubercle bacilli requires functional glyoxylate and methylcitrate cycles, respectively. Enzymes involved in both pathways are essential for M. tuberculosis viability and persistence during growth on fatty acids. However, little is known about regulatory factors responsible for adjusting the expression of genes encoding these enzymes to particular growth conditions. Here, we characterized the novel role of PrpR as a transcription factor that is directly involved in regulating genes encoding the key enzymes of methylcitrate (methylcitrate dehydratase [PrpD] and methylcitrate synthase [PrpC]) and glyoxylate (isocitrate lyase [Icl1]) cycles. Using cell-free systems and intact cells, we demonstrated an interaction of PrpR protein with prpDC and icl1 promoter regions and identified a consensus sequence recognized by PrpR. Moreover, we showed that an M. tuberculosis prpR-deletion strain exhibits impaired growth in vitro on propionate as the sole carbon source. Real-time quantitative reverse transcription-polymerase chain reaction confirmed that PrpR acts as a transcriptional activator of prpDC and icl1 genes when propionate is the main carbon source. Similar results were also obtained for a non-pathogenic Mycobacterium smegmatis strain. Additionally, we found that ramB, a prpR paralog that controls the glyoxylate cycle, is negatively regulated by PrpR. Our data demonstrate that PrpR is essential for the utilization of odd-chain-length fatty acids by tubercle bacilli. Since PrpR also acts as a ramB repressor, our findings suggest that it plays a key role in regulating expression of enzymes involved in both glyoxylate and methylcitrate pathways.

Summary RNA-binding proteins (RBPs) commonly feature multiple RNA-binding domains (RBDs), which provide these proteins with a modular architecture. Accumulating evidence supports that RBP architectural modularity and adaptability define the specificity of their interactions with RNA. However, how multiple RBDs recognize their cognate single-stranded RNA (ssRNA) sequences in concert remains poorly understood. Here, we use Upstream of N-Ras (Unr) as a model system to address this question. Although reported to contain five ssRNA-binding cold-shock domains (CSDs), we demonstrate that Unr includes an additional four CSDs that do not bind RNA (pseudo-RBDs) but are involved in mediating RNA tertiary structure specificity by reducing the conformational heterogeneity of Unr. Disrupting the interactions between canonical and non-canonical CSDs impacts RNA binding, Unr-mediated translation regulation, and the Unr-dependent RNA interactome. Taken together, our studies reveal a new paradigm in protein-RNA recognition, where interactions between RBDs and pseudo-RBDs select RNA tertiary structures, influence RNP assembly, and define target specificity.