Protein splicing in the absence of an intein penultimate histidine

Protein splicing is a self-catalytic process in which an intervening sequen ce, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitat es this reaction by assisting in Asn cyclization, which results in C-termin al splice junction cleavage. However, many inteins do not have a penultimat e His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins wit hout penultimate His residues was examined. Both the Methanococcus jannasch ii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins splic ed. Splicing of the phosphoenolpyruvate synthase intein improved when its p enultimate Phe was changed to His, but splicing of the RNA polymerase subun it 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 a ncestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that sel ective pressure for efficient expression of the RNA polymerase yielded an i ntein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.