We have been investigating the regulation of expression of SecA, a cytoplasmic ATPase that plays a major role in the translocation of proteins through the SecYEG complex in E. coli. SecA expression is regulated at the translational level by secM, a gene that resides immediately upstream of secA in the same operon and that encodes a presecretory protein. SecM contains a 17 amino acid C-terminal sequence motif (150FXXXXWIXXXXGIRAGP166) that normally induces a transient translation arrest. Under conditions of secretion stress, however, the membrane targeting of SecM is inhibited. This targeting defect prolongs translation arrest and increases SecA synthesis by concomitantly altering the structure of the secM-secA mRNA. Translation arrest requires the recognition of the SecM C-terminal motif inside the ribosome tunnel, but the mechanism of recognition is unknown. While single mutations in the motif impair recognition, we found that novel arrest-inducing peptides can be created through remodeling of the SecM C-terminus. We found that R163 is indispensable, but that flanking residues that vary in number, position, and side chain chemistry play an important secondary role in translation arrest. The observation that individual SecM variants show a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on our results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intra-tunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the ribosome tunnel and help explain the striking diversity of arrest-inducing peptides found in bacteria, fungi and higher eukaryotes. We have also found that the SecM signal peptide plays an essential role in this regulatory process by acting as a molecular timer that coordinates membrane targeting with the synthesis of the arrest motif. We found that signal peptide mutations that alter targeting kinetics and insertions or deletions that change the distance between the SecM signal peptide and the arrest motif perturb the balance between the onset and release of arrest that is required to regulate SecA synthesis properly. Furthermore, we found that the strength of the interaction between the ribosome and the SecM arrest motif is calibrated to ensure the release of arrest upon membrane targeting. Our results strongly suggest that several distinctive features of the SecM protein evolved as a consequence of constraints imposed by the ribosome and the Sec machinery. In a recent effort to identify novel translation arrest phenomena in E. coli, we compared the proteome of a wild-type strain to a strain that has a mutation in the ribosomal L22 subunit that appears to exert a broad inhibitory effect on peptide-mediated translation arrest. By impairing SecM-mediated translation arrest, the mutation reduces the steady-state level of SecA. While the mutation does not affect the level of most proteins, it dramatically reduces the level of antigen 43 (Ag43), a secreted virulence factor that promotes biofilm formation. We found, however, that the decrease in Ag43 concentration was an indirect effect of the reduction in secA expression and was not due to a defect in the recognition of a novel translation arrest-inducing peptide. Our results suggest that peptide-mediated translation arrest is used relatively infrequently to regulate gene expression in E. coli.
|Yap, Mee-Ngan F; Bernstein, Harris D (2013) Mutations in the Escherichia coli ribosomal protein L22 selectively suppress the expression of a secreted bacterial virulence factor. J Bacteriol 195:2991-9|
|Yap, Mee-Ngan; Bernstein, Harris D (2009) The plasticity of a translation arrest motif yields insights into nascent polypeptide recognition inside the ribosome tunnel. Mol Cell 34:201-11|