Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction

Kawasaki, Masato; Satow, Yoshinori; Ohya, Yoshikazu; Anraku, Yasuhiro

doi:10.1016/s0014-5793(97)00850-8

Cited by 19 publications

(21 citation statements)

References 16 publications

(44 reference statements)

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“…A third in vitro splicing system, also involving the Sce VMA intein, was developed by Anraku and coworkers (19) and is based on the fact that expression of fusion proteins containing this intein in E. coli led to the accumulation of unspliced precursor as inclusion bodies, which could be solubilized in 6 M guanidine hydrochloride and made to undergo protein splicing by dialysis. Preliminary mechanistic studies by using this experimental system demonstrated that protein splicing is an intramolecular process, which is resistant to protease inhibitors (20); however, a serious limitation of these studies is that the refolding process and protein splicing were not clearly separated.…”

Section: Resultsmentioning

confidence: 99%

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

Mills

Lew

Jiang

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

145

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Protein splicing involves the self-catalyzed excision of protein splicing elements, or inteins, from f lanking polypeptide sequences, or exteins, leading to the formation of new proteins in which the exteins are linked directly by a peptide bond. To study the enzymology of this interesting process we have expressed and purified N-and C-terminal segments of the Mycobacterium tuberculosis RecA intein, each Ϸ100 amino acids long, fused to appropriate exteins. These fragments were reconstituted into a functional protein splicing element by renaturation from 6 M urea. When renaturation was carried out in the absence of thiols, the reconstituted splicing element accumulated as an inactive disulfide-linked complex of the two intein fragments, which could be induced to undergo protein splicing by reduction of the disulfide bond. This provided a useful tool for separately investigating the requirements for the reconstitution of the intein fragments to yield a functional protein splicing element and for the protein splicing process per se. For example, the pH dependence of these processes was quite different, with reconstitution being most efficient at pH 8.5 and splicing most rapid at pH 7.0. The availability of such an in vitro protein splicing system opens the way for the exploration of intein structure and the unusual enzymology of protein splicing. In addition, this trans-splicing system is a potential protein ligase that can link any two polypeptides fused to the N-and C-terminal intein segments.Protein splicing is an unusual process by which the flow of information from a gene to its protein product is modulated posttranslationally so as to yield two functionally unrelated proteins. It involves the precise, self-catalyzed excision of an intervening polypeptide sequence, the intein, from an inactive precursor protein with the concomitant joining of the flanking sequences, the exteins, to produce a new functional protein (Fig. 1). All information and catalytic groups required for protein splicing reside in the intein and the two flanking amino acids. With the elucidation of the chemical mechanism of protein splicing (for review see refs. 1 and 2), it has become clear that inteins constitute a class of highly unusual enzymes: (i) they catalyze three mechanistically distinct reactions, two of which, acyl rearrangement of a peptide bond adjacent to cysteine or serine (3, 4) and cyclization of asparagine coupled to peptide bond cleavage (5, 6), have also been found to occur naturally in polypeptides, but only under extreme conditions or at very slow rates; (ii) they act on amino acid residues at their own N and C termini, so that the intein enzymes are also their own substrate, analogous to the role of catalytic RNA in the self-splicing of group I introns (7); and (iii) they catalyze a transesterification reaction between their N and C termini, and their catalytic center therefore comprises both extremities of the polypeptide chain, a situation that is rarely encountered in conventional enzymes and suggests an un...

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Section: Resultsmentioning

confidence: 99%

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

Mills

Lew

Jiang

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

145

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“…The protein splicing domain of the intein is formed from the regions Xanking the endonuclease domain and the two domains have little or no functional or structural overlap (Duan et al, 1997;Moure et al, 2002). The chemical steps in protein splicing that generate the processed VMA have been described (Chong et al, 1996;Kawasaki et al, 1997) and further structural studies have added to these data (Duan et al, 1997;Moure et al, 2002;Anraku et al, 2005). created artiWcial, splicing-competent, VMA mini-inteins by the deletion of the internal homing endonuclease region (motifs C, D, E and H) from the S. cerevisiae VMA intein.…”

Section: The Vma Intein Of Saccharomycetesmentioning

confidence: 99%

The nuclear-encoded inteins of fungi

Poulter

Goodwin

Butler

2007

Fungal Genetics and Biology

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“…Protein splicing proceeds by an intramolecular pathway requiring no exogenous cofactors. [4,5] All of the information necessary for splicing resides within the intein polypeptide plus the first extein amino acid at the C-terminal splice junction.…”

Section: Introductionmentioning

confidence: 99%

Dissecting the Chemistry of Protein Splicing and Its Applications

Noren

Wang

Perler³

2000

Angewandte Chemie International Edition

251

114

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Protein splicing, the protein equivalent of RNA splicing, is a newly discovered posttranslational process that proceeds through a branched protein intermediate and produces two separate polypeptides from one gene. The experimental data used to distinguish among the proposed protein-splicing mechanisms are presented along with the progress made towards fully describing the mechanism. Numerous protein engineering applications using modified inteins have been developed, including the generation of alpha-thioesters in proteins, which circumvent the limits of solid-phase peptide synthesis.

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Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction

Cited by 19 publications

References 16 publications

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

Protein splicing in trans by purified N- and C-terminal fragments of the Mycobacterium tuberculosis RecA intein

The nuclear-encoded inteins of fungi

Dissecting the Chemistry of Protein Splicing and Its Applications

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