title: Global profiling of SRP interaction with nascent polypeptides.
abstract: Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.
authors.forename: Daniela
authors.initials: D
authors.lastname: Schibich
authors.forename: Felix
authors.initials: F
authors.lastname: Gloge
authors.forename: Ina
authors.initials: I
authors.lastname: Pöhner
authors.forename: Patrik
authors.initials: P
authors.lastname: Björkholm
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Wade
authors.forename: Gunnar
authors.initials: G
authors.lastname: von Heijne
authors.forename: Bernd
authors.initials: B
authors.lastname: Bukau
authors.forename: Günter
authors.initials: G
authors.lastname: Kramer
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020543:Proteome / Q000096:biosynthesis / Q000378:metabolism*; D040901:Proteomics; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D018271:Signal Recognition Particle / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; D012329:RNA, Bacterial; D012333:RNA, Messenger; D018271:Signal Recognition Particle; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase