UCSF

During infection, Mycobacterium tuberculosis delivers critical ESX-1 virulence factors that modulate host cell function and stimulate adaptive immunity. Precise control of ESX-1 secretion may be essential for determining M. tuberculosis pathogenesis. Here we report the crystal structure for EspR, a key transcriptional regulator of ESX-1 secretion. The helix-turn-helix (HTH) domains of EspR are arranged in an unusual conformation in which they are splayed at an oblique angle to each other, suggesting that EspR binds DNA in a profoundly different way than most other known HTH regulators. By mapping the EspR binding sites in the espACD promoter, using both in vivo and in vitro binding assays, we showed that the EspR operators are located unusually far from the promoter. The EspR dimer binds to these sites cooperatively, but the two "half-sites" contacted by each DNA recognition motif are separated by 177 base pairs. The distinctive structure of EspR and the exceptional arrangement of its operator contacts suggest that it could promote DNA looping in its target promoter. Here we present multiple pieces of data that, taken together, support the direct looping model. With a transcription reporter assay, we showed that EspR activity is DNA phase-dependent, consistent with looping between its sites. Using ChIP-chip, we determined the global binding landscape of EspR, which includes regions important for ESX-1 secretion, PDIM synthesis, and control of its own promoter. Through independent in vivo and in vitro methods, we identified the EspR consensus binding site, and find that its distribution and spacing throughout the genome is consistent with EspR-mediated looping as a widespread phenomenon. Finally, we found a key mechanistic clue by concurrent studies of another transcriptional regulator, Lsr2, which appears to be epistatic to EspR for espACD transcription. By comparing the genome-wide binding sites and determining the complete transcriptional regulons of EspR and Lsr2, we found that the function of these two factors is linked at many sites throughout the genome. We discuss potential mechanisms for global co-regulation by EspR and Lsr2.