The C-terminal amino acid sequence of nascent peptide is a major determinant of SsrA tagging at all three stop codons.
Recent studies on endogenous SsrA-tagged proteins have revealed that the tagging could occur at a position corresponding to the normal termination codon. During the study of SsrA-mediated Lacl tagging (Abo et al., EMBO J, 2000 19:3762-3769), we found that a variant Lacl (Lacl deltaC1) lacking the last C-terminal amino acid residue is efficiently tagged in a stop codon-dependent manner. SsrA tagging of Lacl deltaC1 occurred efficiently without Lacl binding to the lac operators at any one of three stop codons. The C-terminal (R)LESG peptide of Lacl deltaC1 was shown to trigger the SsrA tagging of an unrelated protein (CRP) when fused to its C terminus. Mass spectrometry analysis of the purified fusion proteins revealed that SsrA tagging occurs at a position corresponding to the termination codon. The alteration of the amino acid sequence but not the nucleotide sequence of the C-terminal portion eliminated the tagging. We also showed that the tagging-provoking sequences cause an efficient translational readthrough at UGA but not UAA codons. In addition, we found that C-terminal dipeptides known to induce an efficient translation readthrough could cause an efficient tagging at stop codons. We conclude that the amino acid sequence of nascent polypeptide prior to stop codons is a major determinant for the SsrA tagging at all three stop codons.
Sunohara
Takafumi
T
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan.
Abo
Tatsuhiko
T
Inada
Toshifumi
T
Aiba
Hiroji
H
D000596:Amino Acids / Q000378:metabolism*; D001426:Bacterial Proteins; D015153:Blotting, Western; D018388:Codon, Terminator; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D056968:Lac Repressors; D013058:Mass Spectrometry; D009875:Operator Regions, Genetic; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D001426:Bacterial Proteins; D018388:Codon, Terminator; D002373:Cyclic AMP Receptor Protein; D017931:DNA Primers; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D010455:Peptides; D012329:RNA, Bacterial; D012097:Repressor Proteins; C096222:tmRNA