Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family

Dassa, Bareket; London, Nir; Stoddard, Barry; Schueler‐Furman, Ora; Pietrokovski, Shmuel

doi:10.1093/nar/gkp095

Cited by 88 publications

(117 citation statements)

References 68 publications

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“…We characterized for the first time the functionality, in terms of protein splicing and self-cleavage activities, of four novel hypothetical split inteins, gp41-1, gp41-8, NrdJ-1, and IMPDH-1, previously predicted from metagenomic sequences (27). All pairs of split intein protein fragments were assessed as model fusion proteins under identical extein context to allow their comparison with the previously reported extremely efficient and fast-splicing split intein Npu DnaE (26).…”

Section: Discussionmentioning

confidence: 99%

“…The obvious prerequisites for this behavior are a highly optimized recognition and folding of the split intein fragments. For the association of natively split intein fragments, complementary local charge distributions appear to be an important parameter (7,27,29,42,43). The fragments of the Ssp DnaE inteins are positively and negatively charged and form a complex in solution with a rate approaching the diffusion limit (29), whereas the rate-determining step appears to be an intramolecular rearrangement within the complex.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid Construction-Gene sequences and specific details of novel split inteins gp41-1, gp41-8, NrdJ-1, and IMPDH-1 are already described elsewhere (27). Gene synthesis of split intein fragments was performed by Biomatik (Cambridge, Canada), including flanking restriction sites for easy subcloning into the previously described pAU08 and pVS01 plasmids (26).…”

Section: Methodsmentioning

confidence: 99%

“…Most recently, a search in Global Ocean Sampling (GOS) environmental metagenomic sequence data was performed to find fractured genes that contain novel split inteins (27). These fractured genes code for potentially essential cellular proteins, and in the case of four insertion sites (gp41 DNA helicase, inosine-5Ј-monophosphate dehydrogenase (IMPDH), ribonucleotide reductase catalytic subunit NrdJ, and DnaE polymerase II subunit ␣), the complete loci including the split inteins could be assembled.…”

mentioning

confidence: 99%

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Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Carvajal-Vallejos

Pallisse

Mootz

et al. 2012

Journal of Biological Chemistry

153

186

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Background: Hypothetical natural split inteins from environmental metagenomic sources were evaluated experimentally for the first time. Results: Four inteins showed the highest known reaction rates and efficiencies for protein trans-splicing and could be used efficiently for C-cleavage. Conclusion: These inteins pushed the catalytic limit of protein trans-splicing, although this was unpredictable from their primary sequence. Significance: These inteins hold great potential as versatile protein engineering tools.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Carvajal-Vallejos

Pallisse

Mootz

et al. 2012

Journal of Biological Chemistry

153

186

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“…We have shown here that mobA can be spread by horizontal transmission, and it is likely that other similar constructs will also be shown to practice homing. Recent metagenomic analysis of environmental DNA samples has yielded additional examples of genes split by insertion of putative homing endonucleases (32), indicating that the infectious splitting of essential proteins may be a more common phenomenon than previously thought.…”

Section: Origin Of the Bypassmentioning

confidence: 99%

A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette

Bonocora

Zeng

Abel

et al. 2011

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

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Since its initial description more than two decades ago, the ribosome bypass (or "hop") sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an inphase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a homing endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated homing endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron. bacteriophage gene structure | group I intron | horizontal gene transfer | ribosomal frameshifting | mobile genetic element I n most of the phages of the T4 superfamily, the large subunit of DNA topoisomerase is a single polypeptide encoded by gene 39. However, in phage T4 this gene is disrupted by a 1-kb insertion, creating a truncated gene 39 plus a new gene (gene 60) that encodes the remainder of the topoisomerase subunit (1, 2) ( Fig. 1). Furthermore, the C terminus of the truncated gene 39 and the N terminus of the newly created gene 60 each contain additional amino acid residues (43 and 30, respectively) that were not present in the original gene 39 homologs. These two independently translated proteins interact with the small subunit (encoded by gene 52) to create an enzymatically active topoisomerase (4). A second, 50-nt insertion beginning with an inphase stop codon, is inserted into the newly created downstream gene 60 (2). Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame (5). Features involved in this remarkable process include: the stop codon, the codon at which translation resumes, a portion of the pre-hop nascent peptide, a stem-loop at the start of the bypassed sequence, a Shine/Dalgarno-like sequence within the bypassed RNA, and the structure of the bypassed mRNA (5-8).The genome sequence of phage T4 (GenBank accession no. NC_000866) indicated that the insertion into gene 39 contains two ORFs: an apparent H-N-H homing endonuclease ps...

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Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

et al. 2020

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Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the À1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the À1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.

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Fractured genes: a novel genomic arrangement involving new split inteins and a new homing endonuclease family

Cited by 88 publications

References 68 publications

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

Unprecedented Rates and Efficiencies Revealed for New Natural Split Inteins from Metagenomic Sources

A homing endonuclease and the 50-nt ribosomal bypass sequence of phage T4 constitute a mobile DNA cassette

Substrate specificities of inteins investigated by QuickDrop‐cassette mutagenesis

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