Peptide-Based Investigation of the Escherichia coli RNA Polymerase 蟽70:Core Interface As Target Site

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• 作者：Kristina H眉secken ; Matthias Negri ; Martina Fruth ; Stefan Boettcher ; Rolf W. Hartmann ; Joerg Haupenthal
• 刊名：ACS Chemical Biology
• 出版年：2013
• 出版时间：April 19, 2013
• 年：2013
• 卷：8
• 期：4
• 页码：758-766
• 全文大小：696K
• 年卷期：v.8,no.4(April 19, 2013)
• ISSN：1554-8937

文摘

The number of bacterial strains that are resistant against antibiotics increased dramatically during the past decades. This fact stresses the urgent need for the development of new antibacterial agents with novel modes of action targeting essential enzymes such as RNA polymerase (RNAP). Bacterial RNAP is a large multi-subunit complex consisting of a core enzyme (subunits: 伪₂尾尾鈥蚕? and a dissociable sigma factor (蟽⁷⁰; holo enzyme: 伪₂尾尾鈥蚕壪?sup>70) that is responsible for promoter recognition and transcription initiation. The interface between core RNAP and 蟽⁷⁰ represents a promising binding site. Nevertheless, detailed studies investigating its druggability are rare. Compounds binding to this region could inhibit this protein鈥損rotein interaction and thus holo enzyme formation, resulting in inhibition of transcription initiation. Sixteen peptides covering different regions of the Escherichia coli 蟽⁷⁰:core interface were designed; some of them鈥揳ll derived from 蟽⁷⁰ 2.2 region鈥搇ed to a strong RNAP inhibition. Indeed, an ELISA-based experiment confirmed the most active peptide P07 to inhibit the 蟽⁷⁰:core interaction. Furthermore, an abortive transcription assay revealed that P07 impedes transcription initiation. In order to study the mechanism of action of P07 in more detail, molecular dynamics simulations and a rational amino acid replacement study were performed, leading to the conclusion that P07 binds to the coiled-coil region in 尾鈥?and that its flexible N-terminus inhibits the enzyme by interaction with the 尾鈥?lid-rudder-system (LRS). This work revisits the 尾鈥?coiled-coil as a hot spot for the protein鈥損rotein interaction inhibition and expands it by introduction of the LRS as target site.