MOLECULAR DISSECTION OF THE MYCOBACTERIUM-TUBERCULOSIS RECA INTEIN - DESIGN OF A MINIMAL INTEIN AND OF A TRANSSPLICING SYSTEM INVOLVING 2 INTEIN FRAGMENTS

Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify th e domains of inteins that are essential for protein splicing, the inte in sequence embedded in the recA gene of Mycobacterium tuberculosis wa s genetically dissected. The effect of various modifications of the in tein on the ability to mediate splicing was studied in Escherichia col i transformed with plasmids is which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli ma ltose-binding protein and a polypeptide containing a hexahistidine seq uence as the N- and C-exteins, respectively. One type of genetic alter ation of the RecA intein involved deletion of the the central region e ncoding 229 amino acids (aa), representing the entire homing endonucle ase homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wi ld-type intein, indicating that the homing endonuclease domain plays n o role in the protein-splicing process and that the protein-splicing a ctive center is confined to the Nand C-terminal segments of the intein , less than 110 aa each. Another type of alteration involved the intro duction of overlapping translation termination and initiation codons i n-frame into the intein coding region. The modified RecA intein, altho ugh synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splic ing domains can interact with sufficient affinity and specificity to a llow protein-splicing to occur in trans. The efficiency of trans-splic ing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intei n fragments was only about 110 aa each. (C) 1998 Elsevier Science B.V.