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¹ Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany
² Fakultät für Biologie, Universität Freiburg, D-79104 Freiburg, Germany
³ Institut für Biochemie der Eidgenössischen Technischen Hochschule Zürich, CH-8092 Zürich, Switzerland

Address correspondence to Matthias Müller, Institut für Biochemie und Molekularbiologie, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany. Tel.: 49-761-203-5265. Fax: 49-761-203-5274. email: matthias.mueller{at}biochemie.uni-freiburg.de

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.

Key Words: L23; nascent polypeptide chains; protein targeting; signal recognition particle; Trigger factor

Abbreviations used in this paper: DSS, disuccinimidyl suberate; INV, inside-out, inner membrane vesicle; RNC, ribosome–nascent chain complex; SRP, signal recognition particle; TF, Trigger factor; Tmd-Phe, L-4'-(3-[trifluoromethyl]-3H-diazirin-3-yl) phenylalanine.

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