Abstract
The Vent DNA polymerase gene from Thermococcus litoralis contains two in-frame insertions that must be spliced out to form the mature polymerase. Primer extension and cDNA PCR revealed no evidence of spliced RNA to account for this editing. In contrast, pulse-chase analysis indicated that expression constructs lacking the first insertion produced a protein precursor in Escherichia coli that was processed post-translationally to form polymerase and I-TliI, the endonuclease protein that is the product of the second insertion. At least one intermediate, which migrated more slowly than the precursor and may be branched, was also detected. Amino acid substitutions at the splice junction slowed or blocked the protein splicing reaction. Processing occurs in several heterologous systems, indicating either self-splicing or ubiquitous splicing factors. Processing occurs in a mutant lacking I-TliI endonuclease activity, establishing the independence of splicing and endonuclease activities.