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Conditional protein splicing of inteins with a non‐canonical C‐terminal glutamine
Author(s) -
Nicastri Michael C.,
Xega Kristina,
Reitter Julie N.,
Mills Kenneth V.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.959.1
Subject(s) - intein , protein splicing , rna splicing , peptide bond , biochemistry , homing endonuclease , cleavage (geology) , thioester , chemistry , mutant , biology , peptide , genetics , stereochemistry , microbiology and biotechnology , gene , enzyme , endonuclease , paleontology , rna , fracture (geology)
Inteins are intervening polypeptides that self‐catalyze their own excision from the flanking polypeptides, or exteins, in a process called protein splicing. The reaction begins with an amide‐thioester rearrangement of the peptide bond that links the N‐extein to the intein. Next, the intein either proceeds to splice, or, if downstream steps are prevented, the thioester can be cleaved. We have examined inteins interrupting PolII proteins in both Methanoculleus marisnigri ( Mma ) and Methanospirillum hungatei ( Mhu ). Both inteins have C‐terminal Gln in place of the highly conserved Asn. We have observed that the inteins promote mostly N‐terminal cleavage with the native Gln, but promotes some protein splicing with mutation to the conserved Asn. Also, when expressed under oxidizing conditions, the Mma PolII intein accumulates unspliced precursor, which can be induced to undergo N‐terminal cleavage upon in vitro incubation with 1,4‐dithiothreitol. This suggests that a disulfide bond, perhaps between the terminal Cys residues, could regulate the initiation of protein splicing. This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.