In vitro proteolytic processing of a diubiquitin and a truncated diubiquitin formed from in vitro-generated mRNAs.
Ubiquitin, a highly conserved protein of 76 amino acids found in all eukaryotes, is translated from mRNAs that contain either multiple, contiguous coding sequences of the protein or a single ubiquitin coding sequence fused to sequences coding for 52 or 76 amino acids. We describe here formation of monoubiquitin from in vitro translation of mRNAs containing either two complete sequences or one complete ubiquitin and 60% of a second ubiquitin. No diubiquitin precursor was found with the complete diubiquitin mRNA, but the truncated mRNA formed proteins with apparent molecular masses of 30, 24, 7, and 4 kDa. The latter two are the expected products from truncated ubiquitin mRNA. The 30-kDa protein was immunoprecipitated by anti-ubiquitin antibodies and was converted to ubiquitin and the 4-kDa form by a ubiquitin isopeptidase-like activity in wheat germ. Other data indicated that the 30-kDa protein had multiple ubiquitins, all linked by isopeptide bonds to the truncated ubiquitin. One of these was the radiolabeled translation product, which should have been linked to the truncated protein by a normal peptide bond. A model is proposed in which ubiquitin itself participates in a transpeptidase activity.
Agell
N
N
Department of Microbiology and Immunology, Washington University School of Medicine, Saint Louis, MO 63110.
Bond
U
U
Schlesinger
M J
MJ
D003001:Cloning, Molecular; D066298:In Vitro Techniques; D008970:Molecular Weight; D010458:Peptidyl Transferases / Q000378:metabolism; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014452:Ubiquitins / Q000235:genetics*
D011498:Protein Precursors; D012333:RNA, Messenger; D014452:Ubiquitins; D010458:Peptidyl Transferases