Identification of endogenous SsrA-tagged proteins reveals tagging at positions corresponding to stop codons.
The SsrA.SmpB quality control system adds a C-terminal degradation peptide (AANDENYALAA) to nascent chains on stalled ribosomes, thereby freeing the ribosome and ensuring proteolysis of the tagged protein. An SsrA mutant with the tag sequence AANDEHHHHHH was used to slow degradation and facilitate Ni2+-nitrilotriacetic acid affinity purification. Display of affinity-purified Escherichia coli proteins on two-dimensional gels revealed small quantities of a diverse set of SsrA-H6-tagged proteins, and mass spectroscopy identified LacI repressor, lambda cI repressor, YbeL, GalE, RbsK, and a SlyD-kan(R) fusion protein as members of this set. For lambda repressor and YbeL, the SsrA-H6 tag was added after the natural C terminus of the protein, suggesting that tagging occurred while the ribosome idled at the termination codon of these genes. Potential causes of tagging for the other proteins include interference from translation of downstream reading frames, rare codons, and gene disruption. These and previous results support a broad role for the SsrA.SmpB system in freeing stalled ribosomes and in directing degradation of the products of these frustrated protein synthesis reactions.
Roche
E D
ED
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Sauer
R T
RT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D018388:Codon, Terminator; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics*; D016622:Silver Staining; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D001426:Bacterial Proteins; D018388:Codon, Terminator; D008565:Membrane Proteins; D010455:Peptides; D012329:RNA, Bacterial; C092408:smpB protein, Staphylococcus hominis; C096222:tmRNA