Molecular Genetics

In a process analogous to RNA splicing, some proteins are processed by removal of a polypeptide segment from the middle of the polypeptide chain.

Facts

• The S. cerevisiae TFP1 gene codes for the 69 kDa catalytic subunit of the vacuolar H+ ATPase. The gene's nucleotide sequence has an open reading frame for a 119 kDa polypeptide.
• An amino acid sequence 75% similar to that of the Neurospora crassa catalytic subunit is interrupted by 454 residues of unrelated sequence.
• Precise deletion of the codons for this unrelated sequence results in a gene that produces a fully functional catalytic subunit of the right size.
• Northern blot analysis reveals no evidence of a spliced mRNA.
• The 69 kDa polypeptide is not produced from a wild type gene in which a stop codon has been introduced in the central 454 codon segment.

• A similar extra sequence, called an intein, was found in the pol open reading frame of Thermococcus literalis. A similar intein was later identified in the corresponding open reading frame of Pyrococcus sp. These are thermophilic archaebacteria.
• A gene for a double fusion protein was constructed from genes for the maltose binding protein (MBP), the pol-intein, and paramyosin.
• The double fusion protein was produced in E. coli and could be isolated from it.
• Under controlled conditions (including elevated temperature), the double fusion protein cleaved in vitro to release the pol intein domain and fuse the MBP and paramyosin domains.
• During the cleavage reaction, an intermediate was observed that had two N-termini, those of MBP and of pol.

Interpretations

• Polypeptides synthesized from certain genes contain intervening amino acid sequences known as protein introns or inteins.
• Inteins are released by splicing of the N-terminal exein to the C-terminal exein.
• The information for protein splicing is contained within the intein sequence. The exteins contribute little, if any, to the cleavage reaction.
• Intein removal proceeds via a branched intermediate much as does RNA splicing.

Further information

• Over 140 inteins are now known. They are found in a variety of genes in a variety of organisms.
• Parts of the nucleic acid for some inteins resemble parasitic group I RNA introns. The encoded domain is a site-specific endonuclease that plays a role in transposition of the intein DNA.
• Extein splicing occurs in four steps involving sulfhydryl- or hydroxyl groups on amino acids on the C-terminal side of the split bonds and an asparagine at the C-terminus of the intein (ref). In addition to the intein, the first C-terminal extein residue is required and is the nucleophile for two of the four steps.

• In Synechocystis, a blue-green alga of known genome sequence, inteins interrupt each of 4 genes (dnaE, dnaB, dnaX, gyrB). The dnaE gene is split in two parts by 745 kbp of DNA. One part includes code for the N-terminal part of an intein, while the other (oriented oppositely) programs synthesis of the C-terminal. E. coli transformed with the split gene produces the intact protein. Protein splicing must occur in trans in this case (ref).

This is page 2512 of Molecular Genetics by Ulrich Melcher, © 1997, 1999, 2000, 2003