Identification of a hyperactive variant of the SecM motif involved in ribosomal arrest

Recent studies in several organisms have shown that certain nascent sticky peptides stall in the ribosome during their own translation. Amino acid sequences present at the C-terminal part of Escherichia coli SecM ((150)FSTPVWISQAQGIRAGP(166)) have a well-characterized role in ribosome stalling. To investigate the determinants of the SecM motif responsible for ribosome stalling, we performed a genetic screen for mutants with an altered SecM motif that resulted in altered ribosome stalling. To do this, we used a cat fusion construct containing the SecM motif and a myc-tag (cat'-'myc-secM). This construct expresses cat'-'myc-secM mRNA transcripts predominantly translated by a subset of ribosomes called specialized ribosomes that recognize an altered ribosome binding sequence in the mRNA. While all of the isolated mutants containing mutations at the functionally conserved amino acid residues at positions between 161 and 166 showed decreased ribosome stalling, one mutant sequence containing an amino acid substitution from serine to lysine at position 157 (S157K) showed enhanced ribosome stalling that consequently increased mRNA cleavage. Our results reveal that a functionally not conserved amino acid residue at position 157 of SecM can also affect ribosome stalling and provide additional insight into the molecular mechanisms underlying sticky-peptide-induced ribosome arrest.