Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Xu, Ming‐Qun; Comb, Donald G.; Paulus, Henry; Noren, Christopher J.; Shao, Yang; Perler, Francine B.

doi:10.1002/j.1460-2075.1994.tb06888.x

Cited by 115 publications

(149 citation statements)

References 24 publications

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“…Recently, Chong et al (21) also reported that a branched intermediate accumulated in vivo using the N737A/ C738S mutant of VDE. Our results are consistent with the succinimide-mediated cleavage model at the C-terminal junction (20).…”

Section: Discussionsupporting

confidence: 92%

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Identification of Three Core Regions Essential for Protein Splicing of the Yeast Vma1 Protozyme

Kawasaki¹,

Nogami²,

Satow³

et al. 1997

Journal of Biological Chemistry

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The translation product of the VMA1 gene of Saccharomyces cerevisiae undergoes protein splicing, in which the intervening region is autocatalytically excised and the franking regions are ligated. The splicing reaction is catalyzed essentially by the in-frame insert, VMA1-derived endonuclease (VDE), which is a site-specific endonuclease to mediate gene homing. Previous mutational analysis of the splicing reaction has been concentrated extensively upon the splice junctions. However, it still remains unknown which amino acid residues are crucial for the splicing reaction within the entire region of VDE and its neighboring elements. In this work, a polymerase chain reaction-based random mutagenesis strategy was used to identify such residues throughout the overall intervening sequence of the VMA1 gene. Splicing-defective mutant proteins were initially screened using a bacterial expression system and then analyzed further in yeast cells. Mutations were mapped at the N-and C-terminal splice junctions and around the N-terminal one-third of VDE. We identified four potent mutants that yielded aberrant products with molecular masses of 200, 90, and 80 kDa. We suggest that the conserved His 362 , newly identified as the essential residue for the splicing reaction, contributes to the first cleavage at the N-terminal junction, whereas His 736 assists the second cleavage by Asn cyclization at the C-terminal junction. Mutations in these regions did not appear to destroy the endonuclease activity of VDE.Protein splicing is a posttranslational process, in which an intervening protein sequence is autocatalytically removed from a precursor protein and the two flanking sequences are ligated (1). In the budding yeast Saccharomyces cerevisiae, the nascent 120-kDa VMA1 translational product (Vma1 protozyme (1)) catalyzes protein splicing to yield a 70-kDa catalytic subunit of the vacuolar H ϩ -ATPase and a 50-kDa site-specific endonuclease (VMA1-derived endonuclease; VDE) 1 (2, 3). Since the discovery of protein splicing in S. cerevisiae (2, 3), this compelling reaction has been found in a number of protozymes in eucarya (2-4), bacteria (5-8), and archaea (9, 10).The VDE region in the Vma1 protozyme plays a central role for the protein splicing reaction (11,12). We have also shown that VDE bracketed by only 6 proximal and 4 distal amino acids is autocatalytically processed in vitro (13). These results support the idea that the VDE sequence and a few external amino acids 2 contain sufficient information for protein splicing.Only short amino acid sequence motifs are conserved among protein splicing elements (7). Thiol-or hydroxyl-containing Cys, Ser, or Thr is found at both splice junctions. A few hydrophobic amino acids are present in front of an invariant His-Asn dipeptide at the C-terminal splice junction. These residues around the splice sites have been found to play key roles in the protein splicing reaction, based mostly on site-directed mutagenesis studies (5,10,11,12).To understand a mechanism and structural integrity for protei...

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Section: Discussionsupporting

confidence: 92%

“…C-terminal Splice Junction-Asn 737 at the C-terminal junction plays a critical role in protein splicing to form a succinimide ring (20). Cooper et al (12) reported that substitutions of Asn 737 to seven other amino acids (Lys, Ala, Tyr, Gln, Glu, His, and Asp) all resulted in nonspliced products.…”

Section: Discussionmentioning

confidence: 99%

Identification of Three Core Regions Essential for Protein Splicing of the Yeast Vma1 Protozyme

Kawasaki¹,

Nogami²,

Satow³

et al. 1997

Journal of Biological Chemistry

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“…When an intein BI accumulates, the (thio)ester linkage is usually alkaline labile at elevated temperatures. 5,[18][19][20] The affinity purified Asn343Ala BI was incubated overnight in vitro at 4 C, room temperature, 30 C or 37 C, pH 6 or 9, and in the presence or absence of 50 mM DTT [ Fig. 5(A) and data not shown].…”

Section: Characterization Of the Branched Intermediate That Accumulatmentioning

confidence: 99%

“…Historically, intein BIs accumulate when the branch point is an ester rather than a thioester, 18,19 so a triple mutant consisting of Cys323Ser, Asn343Ala, and Thrþ1Ala was constructed. A slowly migrating band accumulated in this triple mutant [ Fig.…”

Section: Trapping the Block F Branched Intermediatementioning

confidence: 99%

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Brace¹,

Southworth²,

Tori³

et al. 2010

Protein Science

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Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post-translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C-extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein-mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N-terminal splice junction to form the class specific branched intermediate after which the N-extein is transferred to the side chain of the Ser, Thr, or Cys at the C-terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.

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“…1). Xu et al (1994) demonstrated that the C-terminal end of the VDE protein is Asn737. Finally, Kawasaki et al (1996) determined the junction site of the in vitro spliced recombinant Vma1 protein (peptide E; see Fig.…”

Section: The Vma1 Gene Contains Genetic Information For the Two Protementioning

confidence: 99%

Protein splicing: its chemistry and biology

Anraku

1997

Genes to Cells

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Protein splicing is a chemical reaction in which a spliced intervening polypeptide is excised from a precursor protein and the flanking N-and C-terminal regions are ligated with the peptide bond to produce two mature proteins. This unique autocatalytic reaction was first discovered in the yeast VMA1 protein, a 120 kDa spliced polypeptide encoded by the VMA1 gene of Saccharomyces cerevisiae. The VMA1 protein catalyses a self protein splicing post-translationally to yield the 70 kDa catalytic subunit of the vacuolar H þ -ATPase and the 50 kDa DNA endonuclease. Accumulating evidence has indicated that splicing precursors distribute widely in many organisms covering eukarya, bacteria and archaea. This article argues and summarizes current chemical and biological views on protein splicing.

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Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation.

Cited by 115 publications

References 24 publications

Identification of Three Core Regions Essential for Protein Splicing of the Yeast Vma1 Protozyme

Identification of Three Core Regions Essential for Protein Splicing of the Yeast Vma1 Protozyme

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

Protein splicing: its chemistry and biology

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