Structural, functional, and genetic analysis of sorangicin inhibition of bacterial RNA polymerase.

A combined structural, functional, and genetic approach was used to investigate inhibition of bacterial RNA polymerase (RNAP) by sorangicin (Sor), a macrolide polyether antibiotic. Sor lacks chemical and structural similarity to the ansamycin rifampicin (Rif), an RNAP inhibitor widely used to treat tuberculosis. Nevertheless, structural analysis revealed Sor binds in the same RNAP beta subunit pocket as Rif, with almost complete overlap of RNAP binding determinants, and functional analysis revealed that both antibiotics inhibit transcription by directly blocking the path of the elongating transcript at a length of 2-3 nucleotides. Genetic analysis indicates that Rif binding is extremely sensitive to mutations expected to change the shape of the antibiotic binding pocket, while Sor is not. We suggest that conformational flexibility of Sor, in contrast to the rigid conformation of Rif, allows Sor to adapt to changes in the binding pocket. This has important implications for drug design against rapidly mutating targets.

Crystal structures of the ADP and ATP bound forms of the Bacillus anti-sigma factor SpoIIAB in complex with the anti-anti-sigma SpoIIAA.

Cell type-specific transcription during Bacillus sporulation is established by sigma(F), the activity of which is controlled by a regulatory circuit involving the anti-sigma factor and serine kinase SpoIIAB, and the anti-anti-sigma SpoIIAA. When ATP is present in the nucleotide-binding site of SpoIIAB, SpoIIAA is phosphorylated, followed by dissociation. The nucleotide-binding site of SpoIIAB is left bound to ADP. SpoIIAB(ADP) can bind an unphosphorylated molecule of SpoIIAA as a stable binding partner. Thus, in this circuit, SpoIIAA plays a dual role as a substrate of the SpoIIAB kinase activity, as well as a tight binding inhibitor. Crystal structures of both the pre-phosphorylation complex and the inhibitory complex, SpoIIAB(ATP) and SpoIIAB(ADP) bound to SpoIIAA, respectively, have been determined. The structural differences between the two forms are subtle and confined to interactions with the phosphoryl groups of the nucleotides. The structures reveal details of the SpoIIAA:SpoIIAB interactions and how phosphorylated SpoIIAA dissociates from SpoIIAB(ADP). Finally, the results confirm and expand upon the docking model for SpoIIAA function as an anti-anti-sigma in releasing sigma(F) from SpoIIAB.