Our genetic results establish that Myx interacts with the RNAP switch region (, S1, S2
)--i.e., with the hinge that mediates rotation of the RNAP clamp relative to the remainder of RNAP and, thus, that mediates opening and closing of the RNAP active-center cleft (). Our biochemical results establish that Myx inhibits interactions of RNAP with promoter DNA, establish that Myx inhibits interactions with the promoter DNA segment that enters the RNAP active-center cleft during transcription initiation (i.e., promoter positions −11 to +15), and establish that Myx inhibits interactions with this promoter DNA segment when it is in a double-stranded state (i.e., the state present before and during entry of promoter DNA into the RNAP active-center cleft, during early stages of transcription initiation) but not when it is present in a single-stranded state (i.e., the state present after entry of promoter DNA into the RNAP active-center cleft, during late stages of transcription initiation) (;S3–S12). Our structural results define contacts between Myx and RNAP and indicate that Myx interacts with an RNAP conformational state in which the RNAP clamp and RNAP switch region are in a partly closed, or partly closed to fully closed, conformation (,, S13–S15
). Based on these results, we propose that Myx inhibits transcription by locking the RNAP switch region in one conformation and thereby locking the RNAP clamp in one conformation--a partly closed, or partly closed to fully closed, conformation--thereby preventing opening of the RNAP active-center cleft to permit entry of double-stranded DNA during transcription initiation. According to this proposal, Myx functions essentially by “hinge jamming.”
Our results further indicate that two other antibiotics--the structurally related α-pyrone antibiotic Cor and the structurally unrelated macrocyclic-lactone antibiotic Rip--share the same target as Myx and the same mechanism as Myx (; Figs. S16–S21
The results define a new target and a new mechanism for inhibition of RNAP. The results, further, provide experimental evidence for functional significance of the RNAP switch region, experimental evidence for functional significance of opening and closing of the RNAP clamp, and experimental evidence supporting the hypothesis that the RNAP clamp must open to permit DNA to enter the RNAP active-center cleft during transcription initiation. As such, the results have implications for understanding mechanisms of transcription and mechanisms of transcriptional regulation.
Based on several considerations, we suggest that the RNAP switch region is an exceptionally attractive target for discovery of new broad-spectrum antibacterial therapeutic agents. First, the switch region comprises residues that are highly conserved in both Gram-positive bacterial RNAP and Gram-negative bacterial RNAP (Fig. S2
)--providing a basis for broad-spectrum activity of compounds that function through the switch region. Second, the switch region contains residues that are not conserved, and indeed are radically different, in human RNAP I, RNAP II, and RNAP III (Fig. S2
)--providing a basis for therapeutic selectivity of compounds that function through the switch region. Third, the switch region is distant from the binding site for rifamycins () and from the binding sites for other characterized inhibitors of bacterial RNAP (see Darst et al., 2004
; Chopra, 2007
), providing a basis for absence of cross-resistance with rifamycins () and for absence of cross-resistance with other characterized inhibitors of bacterial RNAP (unpublished). Fourth, the ligand binding site in the switch region comprises a nearly completely enclosed, predominantly hydrophobic, pocket (), providing a basis for efficient “druggability” by multiple chemotypes (; , , S16, S19
), facilitating in silico
rational design of optimized ligands, and facilitating in silico
virtual screening for new ligands.
We point out that the mechanistic role of the RNAP switch region (hinge for movement of domains) and the structural character of the ligand binding site in the RNAP switch region (nearly completely enclosed, predominantly hydrophobic, pocket) are reminiscent of the mechanistic role and structural character of the HIV-1 reverse transcriptase NNRTI site (non-nucleoside reverse-transcriptase inhibitor site; Kohlstaedt et al., 1992
; Tantillo et al., 1994
; Sluis-Cremer et al., 2004
). The HIV-1 reverse transcriptase NNRTI site is druggable by multiple chemotypes and is the target for multiple antiviral agents in current clinical use. We suggest that the RNAP switch region may exhibit similarly high druggability and similarly high utility.
Priorities for basic research include determination of structures of complexes of RNAP with Cor and Rip (to define interactions with the seven-carbon-atom sidechain extension in Cor and to define interactions with the macrocyclic-lactone chemotype of Rip) and determination of effects of switch-region-target inhibitors on RNAP clamp conformation and dynamics in solution (addressable by use of fluorescence resonance energy transfer; A. Chakraborty, Y. Korlann, D. Wang, S. Weiss, and R.H.E., unpublished). Priorities for applied research include cloning and surrogate-host expression of biosynthetic genes for Myx, Cor, and Rip (to overcome an important obstacle to possible clinical application of Myx, Cor, and Rip--namely, the relatively poor fermentation characteristics of the myxobacterial strains that produce these compounds), in silico rational design of optimized switch-region-target inhibitors, and in silico virtual screening and in vitro target-directed high-throughput screening for new switch-region-target inhibitors.