Campbell, Elizabeth A.

DOE OSTI2020

Rewiring the specificity of extracytoplasmic function sigma factors

Significance Bacterial phenotypes require the concerted expression of multiple genes, usually coordinated by a transcriptional regulator. Although the functions of many genes in sequenced bacterial genomes can be inferred, the regulatory networks that coordinate their expression are only known in a few model systems. Using a bioinformatic and experimental approach, we solve the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major class of bacterial regulators. We develop and use a high-stringency pipeline to predict the genes regulated by 67% of ECF σs in >10,000 species, providing a comprehensive look at the role of a broadly distributed family of gene regulatory proteins. This conceptual and computational framework is potentially applicable to other bacterial regulators.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

The antibiotic sorangicin A inhibits promoter DNA unwinding in a Mycobacterium tuberculosis rifampicin-resistant RNA polymerase

Rifampicin (Rif) is a first-line therapeutic used to treat the infectious disease tuberculosis (TB), which is caused by the pathogen Mycobacterium tuberculosis (Mtb). The emergence of Rif-resistant (Rif R ) Mtb presents a need for new antibiotics. Rif targets the enzyme RNA polymerase (RNAP). Sorangicin A (Sor) is an unrelated inhibitor that binds in the Rif-binding pocket of RNAP. Sor inhibits a subset of Rif R RNAPs, including the most prevalent clinical Rif R RNAP substitution found in Mtb infected patients (S456>L of the β subunit). In this paper, we present structural and biochemical data demonstrating that Sor inhibits the wild-type Mtb RNAP by a similar mechanism as Rif: by preventing the translocation of very short RNAs. By contrast, Sor inhibits the RifR S456L enzyme at an earlier step, preventing the transition of a partially unwound promoter DNA intermediate to the fully opened DNA and blocking the template-strand DNA from reaching the active site in the RNAP catalytic center. By defining template-strand blocking as a mechanism for inhibition, we provide a mechanistic drug target in RNAP. Our finding that Sor inhibits the wild-type and mutant RNAPs through different mechanisms prompts future considerations for designing antibiotics against resistant targets. Also, we show that Sor has a better pharmacokinetic profile than Rif, making it a suitable starting molecule to design drugs to be used for the treatment of TB patients with comorbidities who require multiple medications.

59 BASIC BIOLOGICAL SCIENCES↗