Ribosome profiling reveals multiple roles of SecA in cotranslational protein export

SecA, an ATPase known to posttranslationally translocate secretory proteins across the bacterial plasma membrane, also interacts with ribosomes. Here, we used a combination of ribosome profiling methods to investigate the cotranslational actions of SecA in vivo. SecA scans translating ribosomes at the plasma membrane and cotranslationally engages large periplasmic loops on inner membrane proteins. In coordination with the proton motive force, SecA resolves the cytoplasmic accumulation of periplasmic loops during cotranslational protein translocation. SecA also associates with a subset of secretory proteins at an early stage of translation and mediates their cotranslational transport, whereas the chaperone trigger factor (TF) delays SecA engagement on other secretory proteins to impose a posttranslational mode of translocation. The hydrophobicity of signal sequence is a determinant of nascent protein triage between SecA and TF. Our results elucidate the principles of SecA-driven cotranslational protein translocation and reveal a hierarchical network of protein export pathways in bacteria. Overall design: Investigating the SecA-nascent chain interactions in four different strain backgrounds (WT, ?tig, secA?ZnBD, ?secB) and in WT strain upon CCCP treatment. Monitoring cotranslational targeting events by fractionation-coupled ribosome profiling in WT strain with and without CCCP treatment (all in replicates).