In this report, we describe insights into the function of the ribosome tunnel that were obtained through an analysis of an unusual 25 residue N-terminal motif (EspP(1-25) ) associated with the signal peptide of the Escherichia coli EspP protein. It was previously shown that EspP(1-25) inhibits signal peptide recognition by the signal recognition particle, and we now show that fusion of EspP(1-25) to a cytoplasmic protein causes it to aggregate. We obtained two lines of evidence that both of these effects are attributable to the conformation of EspP(1-25) inside the ribosome tunnel. First, we found that mutations in EspP(1-25) that abolished its effects on protein targeting and protein folding altered the cross-linking of short nascent chains to ribosomal components. Second, we found that a mutation in L22 that distorts the tunnel mimicked the effects of the EspP(1-25) mutations on protein biogenesis. Our results provide evidence that the conformation of a polypeptide inside the ribosome tunnel can influence protein folding under physiological conditions and suggest that ribosomal mutations might increase the solubility of at least some aggregation-prone proteins produced in E. coli.
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