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 translocons1. The existence of parallel co- and post-translational transport pathways2, 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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
We thank members of the Bukau laboratory for valuable contributions; C. Gläßer for support with data analysis; and H. Bernstein for providing plasmid pHQ4. Sequencing was done at the Genomics & Proteomics Core (DKFZ) facility. I.P. and R.C.W. acknowledge support from the Klaus Tschira Foundation. This work was supported by research grants from the Deutsche Forschungsgemeinschaft (SFB638 and FOR1805) to G.K. and B.B., a Human Frontier Science Program grant to B.B. and a grant from the Swedish Research Council to G.v.H.
Extended data figures
This file contains additional details about the proteome-wide SeRP data of SRP-nascent chain interactions in E. coli. Read counts of the translatome and the SRP interactome are compared, substrate identification methods are highlighted, quality scores (pearson correlation coefficient) are listed, initial SRP binding sites are given, retargeting and skipping events are indicated and topology information is provided.
This file contains an analysis of the properties of bound and skipped transmembrane domains (TMDs), and signal sequences.
About this article
Plant and Cell Physiology (2019)