Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.

VemP ( protein xport onitoring olypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in and regulates expression of the downstream genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln codon of encounters the P-site in the translating ribosome, resulting in up-regulation of V.SecDF2 production. Although our previous study suggests that many residues in a highly conserved C-terminal 20-residue region of VemP contribute to its elongation arrest, the exact role of each residue remains unclear. Here, we constructed a reporter system to easily and exactly monitor the arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His-Phe) to identify and characterize the arrest motif. Our results show that 15 residues in the conserved region participate in elongation arrest and that multiple interactions between important residues in VemP and in the interior of the exit tunnel contribute to the elongation arrest of VemP. The arrangement of these important residues induced by specific secondary structures in the ribosomal tunnel is critical for the arrest. Pro scanning analysis of the preceding segment (Met-Phe) revealed a minor role of this region in the arrest. Considering these results, we conclude that the arrest motif in VemP is mainly composed of the highly conserved multiple residues in the C-terminal region.