In vitro protein splicing of purified precursor and the identification of a branched intermediate.
Protein splicing is a posttranslational processing event in which an internal polypeptide is excised from a protein precursor and the terminal polypeptides are then ligated together, resulting in the production of two proteins. This report presents direct evidence for protein splicing by demonstrating in vitro splicing of purified precursor that accumulated when the protein splicing element from Pyrococcus DNA polymerase was cloned into a foreign gene. In vitro splicing was temperature and pH dependent. A slowly migrating species exhibited kinetic properties of a splicing intermediate and was shown to be a branched molecule by N-terminal sequencing. The precursor and slowly migrating species were interconvertible in response to pH shifts.
Xu
M Q
MQ
New England Biolabs, Beverly, Massachusetts 01915.
Southworth
M W
MW
Mersha
F B
FB
Hornstra
L J
LJ
Perler
F B
FB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D016384:Consensus Sequence; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature
D001426:Bacterial Proteins; D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D004259:DNA-Directed DNA Polymerase