Molecular dissection of the Mycobacterium tuberculosis RecA intein: design of a minimal intein and of a trans-splicing system involving two intein fragments.
Most protein-splicing elements (inteins) function both as catalysts of protein splicing and as homing endonucleases. In order to identify the domains of inteins that are essential for protein splicing, the intein sequence embedded in the recA gene of Mycobacterium tuberculosis was genetically dissected. The effect of various modifications of the intein on the ability to mediate splicing was studied in Escherichia coli transformed with plasmids in which the coding sequence for the RecA intein was inserted in-frame between coding regions for the E. coli maltose-binding protein and a polypeptide containing a hexahistidine sequence as the N- and C-exteins, respectively. One type of genetic alteration of the RecA intein involved deletion of the central region encoding 229 amino acids (aa), representing the entire homing endonuclease homology domain. The residual intein (211 aa plus an undecapeptide spacer) was able to promote protein splicing as efficiently as the wild-type intein, indicating that the homing endonuclease domain plays no role in the protein-splicing process and that the protein-splicing active center is confined to the N- and C-terminal segments of the intein, less than 110 aa each. Another type of alteration involved the introduction of overlapping translation termination and initiation codons in-frame into the intein coding region. The modified RecA intein, although synthesized as two separate components, could nevertheless mediate protein splicing, indicating that the N- and C-terminal protein-splicing domains can interact with sufficient affinity and specificity to allow protein-splicing to occur in trans. The efficiency of trans-splicing was much enhanced when the homing endonuclease domain was entirely deleted so that the length of the interacting N- and C-terminal intein fragments was only about 110 aa each.
Shingledecker
K
K
Boston Biomedical Research Institute, Boston, MA 02114, USA.
Jiang
S Q
SQ
Paulus
H
H
D001407:Bacillus; D003001:Cloning, Molecular; D018387:Codon, Initiator; D018388:Codon, Terminator; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D019154:Protein Splicing; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D018387:Codon, Initiator; D018388:Codon, Terminator; D011993:Recombinant Fusion Proteins; D011938:Rec A Recombinases; D004720:Endonucleases