Characterization of the role of SecM protein in the regulation of the Escherichia coli secA gene

The translocation of preproteins across the Escherichia coli inner membrane depends on ATP and involves a complex multi-component secretion apparatus encoded by seven sec genes, secA, B, D, E, F, G, and Y. secA appears to be the only sec gene that is under protein secretion-specific regulation. The secA gene is the second gene in the secM-secA operon and it has been observed that secA translation varies over a tenfold range depending on the status of protein secretion in the cell: secA expression is low during protein secretion-proficient conditions and it rises during secretion-limiting conditions. secA basal expression is set by a translational coupling mechanism utilizing the upstream gene, secM, whereby ribosomes translating the distal portion of secM are needed to disrupt an RNA repressor helix (helix II) that normally blocks secA translational initiation. In this study we have shown that this secretion-responsive regulation of secA depends on the secretability of SecM preprotein by the translocon since signal sequence mutations in secM rendered secA expression constitutive and they acted only when linked in cis to secA. In addition, suppressors of signal sequence defects, prlA (secY) strains containing the secM signal sequence mutations showed partial restoration of secA repression. Based on our results we were able to propose a novel (translation pause-arrest) model for the contribution of SecM protein in secA regulation. The model is based on a pause/arrest mechanism, whereby ribosomal pausing distal in secM is needed to open up the secA Shine-Delgarno sequence that is normally occluded in an RNA helix on secM-secA mRNA, thereby allowing secA translation initiation. During a protein secretion block SecM translation arrest leads to secA derepression.; We also showed by genetic studies and by enzymatic assays with secM-phoA fusions that the GUG codon and not the downstream AUG codon is the correct translation start site of secM. With GUG as the start codon the newly proposed secM signal peptide is unusually long for a Gram-negative organism due to its extended n-region (19 residues). Initial characterization of this N-terminal region indicated that certain atypical amino acyl residues within this extended region are critical for proper secA regulation. We found that mutations within the N-region specifically affect the secM translational pause and secA regulation, while those in the H-region affect both SecM secretion as well as translational pausing and secA regulation. In addition, analysis of mutations at the 3′ end of secM that affect secA regulation allowed us to identify a conserved region (codons 163 and 164) that is required to promote the translational pause.; Recent biochemical analysis of this system has confirmed many of the basic features of this model (Nagatogawa and Ito, 2001). In particular, a natural translational pause within the distal portion of secM was demonstrated, and the length of the secM translational pause was shown to depend on the secretion capability of nascent SecM protein: defects in the secM signal peptide or translocon promoted a prolonged arrest of secM translation and resulted in secA derepression.