Intervening sequences in an Archaea DNA polymerase gene.

Perler, Francine B.; Comb, Donald G.; Jack, William E.; Moran, Laurie S.; Qiang, Boqin; Kucera, Rebecca; Benner, Jack S.; Slatko, Barton E.; Nwankwo, Donald O.; Hempstead, S K

doi:10.1073/pnas.89.12.5577

Cited by 176 publications

(92 citation statements)

References 25 publications

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“…It is missing from a few inteins (present work and Perler et al, 1997) and protein splicing was shown for inteins that naturally (Telenti et al, 1997) or artificially (Chong & Xu, 1997) lack this domain. Until now, the domain was only known to include motifs similar to those of dodecapeptide (DOD, LAGLI-DADG) homing endonucleases (Hirata et al, 1990;Perler et al, 1992;Davis et al, 1994;Pietrokovski, 1994Pietrokovski, , 1996aBult et al, 1996;Fsihi et al, 1996). Mutations of the EN motifs affect the activity of the intein endonuclease (Hodges et al, 1992;Gimble & Stephens, 1995).…”

Section: Intein Motifs Can Be Organized In Functional Domainsmentioning

confidence: 99%

“…Different types of homing endonucleases, residing in self-splicing and archaea introns and as free-standing genes, are responsible for the mobility of their genes (Belfort & Roberts, 1997). Together with this similarity in function, inteins also share various sequence motifs with CADs and a family of homing endonucleases (Hirata et al, 1990;Perler et al, 1992;Davis et al, 1994;Pietrokovski, 1994;Koonin, 1995;Burglin, 1996;Fsihi et al, 1996). This work examines the conserved sequence motifs of inteins and CADs and their presence in other proteins.…”

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Modular organization of inteins and C‐terminal autocatalytic domains

1998

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Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.

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Section: Intein Motifs Can Be Organized In Functional Domainsmentioning

confidence: 99%

mentioning

confidence: 99%

Modular organization of inteins and C‐terminal autocatalytic domains

1998

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“…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)(6)(7)(8), and archaea (9,10).…”

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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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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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“…archaebacteria Thermococcus litoralis (Hodges et al, 1992;Perler et al, 1992) and Pyrococcus species strains GB-D and KODl (T. Imanaka, GenBank entry D29671 submission, 1994), and the ppsl open reading frame of Mycobacterium Ieprae (this work). The only apparent common feature of the spliced proteins is the presence of a nucleotide-binding domain near or within their intein integration region (Neff, 1993).…”

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confidence: 99%

Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins

1994

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Inteins (protein introns) are internal portions of protein sequences that are posttranslationally excised while the flanking regions are spliced together, making an additional protein product. Inteins have been found in a number of homologous genes in yeast, mycobacteria, and extreme thermophile archaebacteria. The inteins are probably multifunctional, autocatalyzing their own splicing, and some were also shown to be DNA endonucleases. The splice junction regions and two regions similar to homing endonucleases were thought to be the only common sequence features of inteins.This work analyzed all published intein sequences with recently developed methods for detecting weak, conserved sequence features. The methods complemented each other in the identification and assessment of several patterns characterizing the intein sequences. New intein conserved features are discovered and the known ones are quantitatively described and localized. The general sequence description of all the known inteins is derived from the motifs and their relative positions. The intein sequence description is used to search the sequence databases for intein-like proteins. A sequence region in a mycobacterial open reading frame possessing all of the intein motifs and absent from sequences homologous to both of its flanking sequences is identified as an intein. A newly discovered putative intein in red algae chloroplasts is found not to contain the endonuclease motifs present in all other inteins. The yeast HO endonuclease is found to have an overall intein-like structure and a few viral polyprotein cleavage sites are found to be significantly similar to the inteins amino-end splice junction motif. The intein features described may serve for detection of intein sequences.

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Intervening sequences in an Archaea DNA polymerase gene.

Cited by 176 publications

References 25 publications

Modular organization of inteins and C‐terminal autocatalytic domains

Modular organization of inteins and C‐terminal autocatalytic domains

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

Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins

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