Abstract
We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A (Sal), and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI: http://dx.doi.org/10.7554/eLife.02451.001
Highlights
Salinamide A (Sal; SalA) and salinamide B (SalB) are structurally related bicyclic depsipeptide antibiotics, each consisting of seven amino acids and two non-amino-acid residues (Trischman et al, 1994; Moore et al, 1999; Figure 1A)
As a first step to determine whether the RNA polymerase (RNAP)-inhibitory activity of Sal is responsible for the antibacterial activity of Sal in culture, we assessed whether Sal inhibits RNAP in bacterial cells in culture
The pattern observed for Sal matches the pattern observed for the reference RNAP inhibitor rifampin (Rif; compare red lines and blue lines in Figure 2A; Lancini and Sartori, 1968; Lancini et al, 1969), and matches the pattern expected from first principles for an RNAP inhibitor
Summary
Salinamide A (Sal; SalA) and salinamide B (SalB) are structurally related bicyclic depsipeptide antibiotics, each consisting of seven amino acids and two non-amino-acid residues (Trischman et al, 1994; Moore et al, 1999; Figure 1A). SalA and SalB are produced by Streptomyces sp. CNB-091, a marine bacterium isolated from the surface of the jellyfish Cassiopeia xamachana (Trischman et al, 1994; Moore and Seng, 1998; Moore et al, 1999), and SalA is produced by Streptomyces sp. SalA and SalB exhibit antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens, Enterobacter cloacae and Haemophilus influenzae, but do not exhibit cytotoxicity against mammalian cells (Trischman et al, 1994; Moore et al, 1999; Figure 1B). SalA and SalB inhibit both Gram-positive and Gram-negative bacterial RNA polymerase (RNAP) in vitro, but do not inhibit human RNAP I, II, or III in vitro (Miao et al, 1997; Figure 1C). A total synthesis of SalA has been reported (Tan and Ma, 2008)
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