title: Real-time observation of signal recognition particle binding to actively translating ribosomes.
abstract: The signal recognition particle (SRP) directs translating ribosome-nascent chain complexes (RNCs) that display a signal sequence to protein translocation channels in target membranes. All previous work on the initial step of the targeting reaction, when SRP binds to RNCs, used stalled and non-translating RNCs. This meant that an important dimension of the co-translational process remained unstudied. We apply single-molecule fluorescence measurements to observe directly and in real-time E. coli SRP binding to actively translating RNCs. We show at physiologically relevant SRP concentrations that SRP-RNC association and dissociation rates depend on nascent chain length and the exposure of a functional signal sequence outside the ribosome. Our results resolve a long-standing question: how can a limited, sub-stoichiometric pool of cellular SRP effectively distinguish RNCs displaying a signal sequence from those that are not? The answer is strikingly simple: as originally proposed, SRP only stably engages translating RNCs exposing a functional signal sequence.
authors.affiliation: Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Noriega
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Jin
authors.identifier: http://orcid.org/0000-0002-6634-4397
authors.initials: J
authors.lastname: Chen
authors.affiliation: Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Walter
authors.affiliation: Department of Structural Biology, Stanford University School of Medicine, Stanford, United States.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Puglisi
mesh_terms: D002138:Calibration; D003199:Computer Systems; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D007700:Kinetics; D010442:Peptide Chain Initiation, Translational; D011485:Protein Binding; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D018271:Signal Recognition Particle
keywords: E. coli; biophysics; cell biology; protein targeting; ribosome translation; signal recognition particle; single molecule fluorescence; structural biology