Mechanism of Protein Localization in Eschericia coli

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DESCRIPTION (provided by applicant): Our overall goal is to elucidate the molecular details of protein translocation across biological membranes utilizing Escherichia coli as a facile genetic and biochemical system. We will focus on a central component, SecA ATPase, which interacts with most components of this system, and whose ATPase and membrane integration activities are at the heart of the energetics and mechanism of translocation. Three specific aims are proposed. (1) To understand SecA-translocon interaction and its dynamics sulfhydryl labeling of SecA-Cys proteins in right side-out membrane vesicles will be employed to develop a high resolution membrane topology map of SecA in comparison to its solution structure. A subunit-switching model of SecA action will be tested. (2) To understand the structural basis of signal peptide binding to SecA and its regulation, the interaction of SecA with a labeled signal peptide will be studied and fluorescence resonance energy transfer will be utilized to map this site on the SecA structure, This approach will also be used to follow inter-domain movement in SecA containing appropriate fluorescent labels to test a clamshell model for regulating access to the signal peptide-binding site (3) The secretion-specific regulation of secA by the translational pause in secM will be studied genetically to define the secM translational-arrest peptide and those components of the translation and secretion machineries that regulate pausing. The requirement for translocon "pulling" of nascent translocating SecM as the signal for releasing the secM translational pause will be tested. These studies should lead to a refined picture of SecA structure, biochemistry, mechanism, and its regulation. They should be of broad significance to understand these processes in parallel systems, to engineer such pathways, and to develop novel anti-bacterial agents.