RRC ID 55853
Author Mori H, Sakashita S, Ito J, Ishii E, Akiyama Y.
Title Identification and characterization of a translation arrest motif in VemP by systematic mutational analysis.
Journal J Biol Chem
Abstract VemP ( V ibrio protein export monitoring polypeptide) is a secretory protein comprising 159 amino acid residues, which functions as a secretion monitor in Vibrio and regulates expression of the downstream V.secDF2 genes. When VemP export is compromised, its translation specifically undergoes elongation arrest at the position where the Gln156 codon of vemP 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 in vivo arrest efficiency of VemP. Using this reporter system, we systematically performed a mutational analysis of the 20 residues (His138-Phe157) 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 (Met120-Phe137) 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.
Volume 293(8)
Pages 2915-2926
Published 2018-2-23
DOI 10.1074/jbc.M117.816561
PII M117.816561
PMID 29317498
PMC PMC5827439
MeSH Amino Acid Motifs Amino Acid Substitution Bacterial Proteins / chemistry Bacterial Proteins / genetics Bacterial Proteins / metabolism* Conserved Sequence Gene Deletion Genes, Reporter Kinetics Lac Operon Models, Molecular* Mutagenesis, Site-Directed Mutation* Oligopeptides / genetics Oligopeptides / metabolism Peptide Chain Termination, Translational* Protein Conformation Protein Engineering* Protein Interaction Domains and Motifs Protein Stability Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / metabolism Ribosomes / chemistry Ribosomes / metabolism* Vibrio / metabolism*
IF 4.106
Times Cited 3
Resource
Prokaryotes E. coli JM109