Mode of action of the microbial metabolite GE23077, a novel potent and selective inhibitor of bacterial RNA polymerase

Abstract

GE23077, a novel microbial metabolite recently isolated from Actinomadura sp. culture media, is a potent and selective inhibitor of bacterial RNA polymerase (RNAP). It inhibits Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) RNAPs with IC 50 values (i.e. the concentration at which the enzyme activity is inhibited by 50%) in the 10 )8 M range, whereas it is not active on E. coli DNA polymerase or on eukaryotic (wheat germ) RNAP II (IC 50 values > 10 )4 M in both cases). In spite of its potent activity on purified bacterial RNAPs, GE23077 shows a narrow spectrum of antimicrobial activity on Gram-positive and Gram-negative bacteria. To investigate the molecular basis of this behaviour, the effects of GE23077 on macromolecular biosynthesis were tested in E. coli cells permeabilized under different conditions. The addition of GE23077 to plasmolyzed cells resulted in an immediate and specific inhibition of intracellular RNA biosynthesis, in a dose-response manner, strongly suggesting that cell penetration is the main obstacle for effective antimicrobial activity of the antibiotic. Biochemical studies were also conducted with purified enzymes to obtain further insights into the mode of action of GE23077. Interestingly, the compound displays a behaviour similar to that of rifampicin, an antibiotic structurally unrelated to GE23077: both compounds act at the level of transcription initiation, but not on the r subunit and not on the formation of the promoter DNA-RNAP complex. Tests on different rifampicin-resistant E. coli RNAPs did not show any cross-resistance between the two compounds, indicating distinct binding sites on the target enzyme. In conclusion, GE23077 is an interesting new molecule for future mechanistic studies on bacterial RNAP and for its potential in anti-infective drug discovery.Keywords: antibiotic; cell permeabilization; natural product; rifampicin; transcription initiation. DNA-directed RNA polymerase (EC 2.7.7.6; RNAP) is the central enzyme of bacterial gene expression, responsible for all cellular RNA synthesis [1]. The catalytically competent ÔcoreÕ RNAP consists of five subunits (a 2 bb¢x, with a combined molecular mass of % 400 kDa) and is capable of elongation and termination. The initiation-competent ÔholoÕ RNAP is composed of the core enzyme and of an additional subunit, r, which confers on RNAP the ability to initiate transcription at specific promoter sites [2,3]. After over four decades of intensive research, RNAP is currently the subject of renewed interest and excitement, owing to recent publication of the crystal structures of the core [4] and holo [5,6] enzymes, and of an RNAP-DNA complex [7].The transcription process consists of three main stages: initiation, elongation and termination. Transcription initiation is a multistep process [8] in which holo RNAP specifically binds to promoter DNA at positions )35 and )10 to form an RNAP-promoter closed complex, melts the DNA duplex around the )10 region to yield an RNAPpromoter open complex, and then initiates transc...

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