Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

Astbury Centre for Structural Molecular Biology University of Leeds Leeds United Kingdom

Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic

School of Biochemistry University of Bristol Bristol United Kingdom

School of Biomedical Sciences Faculty of Biological Sciences University of Leeds Leeds United Kingdom

School of Molecular and Cellular Biology Faculty of Biological Sciences University of Leeds Leeds United Kingdom

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Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon

Distortion of the bilayer and dynamics of the BAM complex in lipid nanodiscs

Inter-domain dynamics in the chaperone SurA and multi-site binding to its outer membrane protein clients

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