Protein splicing of the yeast TFP1 intervening protein sequence

Institute of Molecular Biology, University of Oregon, Eugene 97403.
The EMBO Journal (Impact Factor: 10.43). 07/1993; 12(6):2575-83.
Source: PubMed

ABSTRACT Protein splicing is the protein analogue of RNA splicing in which the central portion (spacer) of a protein precursor is excised and the amino- and carboxy-terminal portions of the precursor reconnected. The yeast Tfp1 protein undergoes a rapid protein splicing reaction to yield a spliced 69 kDa polypeptide and an excised 50 kDa spacer protein. We have demonstrated that the 69 kDa species arises by reformation of a bona fide peptide bond. Deletion analyses indicate that only sequences in the central spacer protein of the Tfp1 precursor are critical for the protein splicing reaction. A fusion protein in which only the Tfp1 spacer domain was inserted into an unrelated protein also underwent efficient splicing, demonstrating that all of the information required for protein splicing resides within the spacer domain. Alteration of Tfp1p splice junction residues blocked or kinetically impaired protein splicing. A protein splicing model is presented in which asparagine rearrangement initiates the self-excision of the spacer protein from the Tfp1 precursor. The Tfp1 spacer protein belongs to a new class of intervening sequences that are excised at the protein rather than the RNA level.

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    • "The second step is a transesterification to the thiol or hydroxyl sidechain of the residue immediately following the intein, (position +1) which in consequence is highly conserved as a serine, threonine or cysteine residue [8,14–16]. The intein is released from this branched intermediate by cyclization of the intein's C-terminal asparagine residue and resulting peptide bond cleavage [15] [17] [18]. The influence of context is evident both in the requirement for a cysteine, threonine or serine at the + 1 position and for residues in the N-extein (position − 1, − 2, etc.) com- Biochimica et Biophysica Acta 1774 (2007) 995 – 1001 "
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    ABSTRACT: As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the alpha-complementation peptide abolished the peptide's ability to restore beta-galactosidase activity, while an active variant allowed complementation. This alpha-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a beta-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.
    Biochimica et Biophysica Acta 09/2007; 1774(8):995-1001. DOI:10.1016/j.bbapap.2007.05.015 · 4.66 Impact Factor
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    • "This latter pathway provided a role for the conserved intein N-terminal nucleophile. Subsequent splicing studies demonstrated an absolute requirement for a Ser, Thr or Cys at the N-terminus of every intein tested (Hodges et al., 1992; Cooper et al., 1993; Xu and Perler, 1996; Evans et al., 1999; Mathys et al., 1999; Wood et al., 1999). The presence of an N-terminal splice junction (thio)ester was proven by several biochemical and biophysical approaches (Chong et al., 1996; Shao et al., 1996; Xu and Perler, 1996; Noren et al., 2000). "
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    ABSTRACT: Variations in the intein-mediated protein splicing mechanism are becoming more apparent as polymorphisms in conserved catalytic residues are identified. The conserved Ser or Cys at the intein N-terminus and the conserved intein penultimate His are absent in the KlbA family of inteins. These inteins were predicted to be inactive, since an N-terminal Ala cannot perform the initial reaction of the standard protein splicing pathway to yield the requisite N-terminal splice junction (thio)ester. Despite the presence of an N-terminal Ala and a penultimate Ser, the KlbA inteins splice efficiently using an alternative protein splicing mechanism. In this non-canonical pathway, the C-extein nucleophile attacks a peptide bond at the N-terminal splice junction rather than a (thio)ester bond, alleviating the need to form the initial (thio)ester at the N-terminal splice junction. The remainder of the two pathways is the same: branch resolution by Asn cyclization is followed by an acyl rearrangement to form a native peptide bond between the ligated exteins.
    The EMBO Journal 10/2000; 19(18):5019-26. DOI:10.1093/emboj/19.18.5019 · 10.43 Impact Factor
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    • "3A!. Two published permissive intein modifications, a deletion of 99 bp in the Mtu recA intein ~Davis et al., 1992! and an insertion of 7 aa into the Sce VMA1 intein ~Cooper et al., 1993! are located in the region between motifs C and E ~Fig. 3A!. "
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    ABSTRACT: The DNA repair protein RecA of Mycobacterium tuberculosis contains an intein, a self-splicing protein element. We have employed this Mtu recA intein to create a selection system for successful intein splicing by inserting it into a kanamycin-resistance gene so that functional antibiotic resistance can only be restored upon protein splicing. We then proceeded to develop an ORFTRAP, i.e., a selection system for the cloning of open reading frames (ORFs). The ORFTRAP exploits the self-splicing properties of inteins (which depend on full-length in-frame translation of a precursor protein) by allowing protein splicing to occur when DNA fragments encoding ORFs are inserted into the Mtu recA intein, whereas DNA fragments containing non-ORFs are selected against. Regions of the Mtu recA intein that tolerate the insertion of additional amino acids were identified by Bgl II linker scanning mutagenesis, and a respective construct was chosen as the ORFTRAP. To test the maximum insert size that could be cloned into ORFTRAP, DNA fragments of increasing length from the Listeria monocytogenes hly gene as well as a genomic library of Haemophilus influenzae were inserted and it was found that the longest permissive inserts were 425 bp and 251 bp, respectively. The H. influenzae ORFTRAP library also demonstrated the strength (strong selection power) and weakness (insertion of very small fragments) of the system. Further modifications should make the ORFTRAP useful for protein expression, epitope mapping, and antigen screening.
    Protein Science 04/1999; 8(3):644-53. DOI:10.1110/ps.8.3.644 · 2.85 Impact Factor
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