Protein splicing in the absence of an intein penultimate histidine

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.