General Characteristics of Protein Splicing

Protein biosynthesis was initially thought to be a simple process; the genetic information in DNA is directly copied into messenger RNAs, which in turn direct the biosynthesis of proteins. However, an unexpected discovery was made by two groups independently in 1990 (Hirata et al. 1990; Kane et al. 1990). In Saccharomyces cerevisiae, the nascent 120-kDa translation product of the VMA1 gene autocatalytically excises a 50-kDa site-specific endonucle-ase (VMAl-derived endonuclease; VDE, also called as Pl-Scel) and splices the two external polypeptides (exteins) to form the 70-kDa catalytic subunit of vaculoar H+-ATPase. This discovery led to the conclusion that the post-trans-lational removal of the internal polypeptide (intein) occurs by protein splicing (Kawasaki et al. 1996). Since the initial discovery of the VMA1 intein, more than 170 putative inteins have been identified in eubacteria, archea and eu-karyotic unicellular organisms (see InBase, the Intein registry website at ht-tp://; New England Biolabs). Protein splicing is a multi-step processing event involving excision of an intein segment from a primary translation product with concomitant ligation of the flanking extein sequences. A typical intein segment consists of 400-500 amino acid residues (Fig. 1). It contains six conserved protein-splicing motifs, Nl, N2, N3, N4, CI and C2, as well as a homing endonuclease sequence embedded between motifs N4 and C2 (Pietrokovski 1998). The endonuclease sequence can be deleted from the intein sequence without abolishing protein splicing (Chong and Xu 1997). At the intein-extein junctions, three conserved amino acid residues are directly involved in the protein-splicing reaction (Evans and Xu 2002; David et al. 2004). They include a Ser or Cys at the intein amino terminus (the first amino acid in motif A), an Asn or Gin at the intein carboxy terminus (the last amino acid in motif G), and a Ser, Thr, or Cys at the beginning of the C-extein. While some inteins can be artificially split into two fragments and still retain activity, a functional naturally split intein-coding sequence was found in 1998 by Liu and colleagues (Wu et al. 1998). This intein is associated with the

Splicing Endonuclease Splicing


Splicing Endonuclease Splicing


N1 N2 N3

N4 ÊN1-ÉN4 C2 C1




C-Extein 1



Q S < T 1



C-Extein |

numbers conserved amino acids

N-Exieiri iHra

domains motifs numbers conserved amino acids

Ssp DnaE

N-Exieiri iHra



Fig. 1. Intein domains, motifs and conserved amino acids. Domains Nl, N2, N3, and N4 contain the N-terminal splicing motifs, while domains CI and C2 contain the C-terminal splicing motifs. The first amino acid residue upstream of the intein is numbered -1, and the intein is numbered sequentially beginning with the N-terminal amino acid residue. The first C-extein residue is numbered +1'. Conserved amino acid residues at position +1 and +1' are indicated split dnaE gene in the genome of Synechocystis sp. PCC6803 (Wu et al. 1998). The Ssp DnaE intein can mediate a trans-splicing reaction when fused to foreign proteins. Compared to artificially split inteins where the endonuclease domain is eliminated, the DnaE intein catalyzes the protein trans-splicing reaction with a higher efficiency (Martin et al. 2001). An important feature of protein splicing is the self-catalyzed excision of the intein and ligation of the flanking exteins without any external enzymes. This feature has been used to devise novel split reporter reconstitutions and their various applications.

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