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SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our lab indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photocrosslinked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec targeting sequences. Second, we used a 2D gel approach to analyse radioactively labelled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterised SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or Trigger Factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ~110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognises nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane embedded Sec machinery.
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