Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition

Abstract

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8–4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds–Nα-aroyl-N-aryl-phenylalaninamides (AAPs)–that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

Structural insights into the mycobacteria transcription initiation complex from analysis of X-ray crystal structures

“…In our model of Eσ A , the mycobacterial σ A N with β′i1 cooperate to impede promoter DNA entry into the channel (Fig. 6b) but do not exclude the possibility that one or both inserts could stabilize the DNA once established in the RNAP cleft as proposed by Lin et al 55…”

“…Once ejected from the RNAP active site cleft, the Mtb σ A N is proposed to cooperate with β′i1 to trap the promoter DNA in the cleft, thereby stabilizing RPo. In support of this hypothesis, Lin et al 55. report that deleting σ A N , β′i1, or both destabilizes complexes of the resulting holoenzymes with the ds-fork DNA.…”

“…The Mtb Eσ A /ds-fork complex is remarkably similar to the Msm RPo: superimposing the Mtb Eσ A /ds-fork complex (PDB ID 5UHA)55 with the Msm RPo results in a r.m.s.d. of 1.02 Å over 2,783 α-carbon positions.…”

“…proposed a different mechanistic role for these structural elements. Lin et al 55. suggest that in the Mtb Eσ A , the σ A N-terminal extension resides in the RNAP active site cleft and is displaced by the entering promoter DNA in a manner analagous to Eco σ 70 1.1 (refs 6, 47).…”

“…The ds-fork promoter template studied by Lin et al 55. is ‘pre-melted’ and lacks DNA upstream of its single-stranded −10 element.…”