The phage T4 nrdB intron: a deletion mutant of a version found in the wild.

Eddy, Sean R.; Gold, Lawrence M.

doi:10.1101/gad.5.6.1032

Cited by 71 publications

(60 citation statements)

References 52 publications

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“…Consistent with this prediction, many phage-and bacterial-encoded RNR genes are interrupted by self-splicing introns or inteins, with the insertion sites often near the active site of the enzymes (16)(17)(18). In phages T4 and RB3, the nrdB gene is interrupted by a group I intron encoding a HNH family homing endonuclease, I-TevIII (19)(20)(21). Of relevance to this study is the free-standing HNH endonuclease, mobE, that is inserted between the nrdA and nrdB genes of phages T4, RB2, RB3, RB15, and LZ7 (9,22) (Fig.…”

mentioning

confidence: 75%

Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

Friedrich

Torrents

Gibb

et al. 2007

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

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In bacterial and phage genomes, coding regions are sometimes interrupted by self-splicing introns or inteins, which can encode mobility-promoting homing endonucleases. Homing endonuclease genes are also found free-standing (not intron-or intein-encoded) in phage genomes where they are inserted in intergenic regions. One example is the HNH family endonuclease, mobE, inserted between the large (nrdA) and small (nrdB) subunit genes of aerobic ribonucleotide reductase (RNR) of T-even phages T4, RB2, RB3, RB15, and LZ7. Here, we describe an insertion of mobE into the nrdA gene of Aeromonas hydrophila phage Aeh1. The insertion creates a unique genes-in-pieces arrangement, where nrdA is split into two independent genes, nrdA-a and nrdA-b, each encoding cysteine residues that correspond to the active-site residues of uninterrupted NrdA proteins. Remarkably, the mobE insertion does not inactivate NrdA function, although the insertion is not a self-splicing intron or intein. We copurified the NrdA-a, NrdA-b, and NrdB proteins as complex from Aeh1-infected cells and also showed that a reconstituted complex has RNR activity. Class I RNR activity in phage Aeh1 is thus assembled from separate proteins that interact to form a composite active site, demonstrating that the mobE insertion is phenotypically neutral in that its presence as an intervening sequence does not disrupt the function of the surrounding gene.bacteriophage Aeh1 ͉ gene structure ͉ intervening sequence H oming endonucleases are a distinctive class of site-specific DNA endonucleases that promote the lateral transfer of their own coding region and flanking DNA between genomes by a recombination-dependent process termed homing (reviewed in ref. 1). Homing endonucleases are often encoded within self-splicing introns and inteins (2-4), but many bacterial and phage genomes possess a significant number of so-called freestanding homing endonucleases that are not encoded within introns or inteins (5, 6). Free-standing endonucleases do not have the benefit of a self-splicing element to minimize their impact on host gene structure and function and, thus, are found at genomic insertion sites that are of low impact, such as intergenic regions. In T4-like phages that infect Escherichia coli and related bacteria, free-standing endonucleases are more abundant than intron-encoded versions (6-9) and promote their spread to phage genomes lacking the endonuclease by an intronless homing pathway (10-12).As a consequence of their abundance in T4-like phages, free-standing endonucleases represent a significant source of genetic variation by promoting recombination between genomes. Many characterized homing endonucleases have recognition sites that lie in genes that function in DNA metabolism, including those that encode aerobic and anaerobic ribonucleotide reductases (RNRs) (13). Three classes of RNRs have been described to date, based on required metallocofactors used to generate a radical intermediate and on structural differences (14). Prokaryotic class Ia aerobic enzymes, typifi...

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confidence: 75%

Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

Friedrich

Torrents

Gibb

et al. 2007

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

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“…A 40-pL aliquot was then boiled for 3 min, and 25 pmol of each primer added, together with 15 nmol of each dNTP, 5 pL of 1OX MgZfcontaining Taq buffer, and 2.5 U Ampli Taq DNA polymerase (Perkin Elmer Co). Forty-five cycles of 94 "C for 1 min; 50 "C for 1 min, and 72 "C for 4 min were performed with a reaction volume of 50 pL, using a modification of the procedure of Eddy and Gold (1991). If a nonpurified phage stock (>lo'' pfu/mL) was used as template, 1-2 p L was used in place of the single plaque.…”

Section: Pcr Techniquesmentioning

confidence: 99%

Phage display of intact domains at high copy number: A system based on SOC, the small outer capsid protein of bacteriophage T4

et al. 1996

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Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-I (HIV-I) envelope glycoprotein; and (3) poliovirus VPl capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to -I O 3 copies per capsid and >IO4 copies per polyhead of V3-sized domains. Phage displaying SOC-VPI were isolated from a 1:106 mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.Keywords: bacteriophage T4; capsid assembly; human immunodeficiency virus antigens; phage display; protein engineering Filamentous phage-based display systems (Smith, 1985) have found widespread use in molecular biology, including many immunologic applications such as antigen presentation and the immunoisolation of desired recombinants (biopanning) (Marks et al., 1992;Smith et al ., 1993;Williamson et al., 1993). However, with filamentous phages, peptides that may be displayed from the major coat protein are limited in size to 6-10 amino acid residues (Kishchenko et al., 1994;Iannolo et al., 1995), although somewhat

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“…This domain will be referred to as the EX,HH-HX3H domain, after the 2 most conserved motifs it contains. The EXIHH-HX3H domain is likely a conserved core of DNase, since this is a large portion of bacteriocin DNases (Schaller & Nomura, 1976), and a part of a fragment of the RBNBi, which is indispensable for enzymatic activity of the intronic DNase (Eddy & Gold, 1991). DNases of the new family can or could mediate a number of processes, including restricting alien DNA (McrA restrictase, bacteriocins, phage T4 ORFs) and conferring mobility to genetic elements (intronic sequences, nifD locus).…”

Section: T P K -R H L D I F I R Y C O N S C N S U S L I]) ~~Y F~k Imentioning

confidence: 99%

“…I ) between agt for glucosyltransferase and 55 for protein necessary for late RNA synthesis; Ytop, composite ORF overlapping 3'-end of topB for large chain of the type I1 topoisomerase. All but 3 sequences were from EMBL (EM), SWISS-PROT (SW), or GENBANK (GB) databases, and the corresponding accession numbers are shown rightmost; 1, Eddy and Gold (1991); 2, Ferat and Michel (1993). Three gaps of variable length were arbitrarily introduced in mt sequences to align Zn-finger and "His''-rich motifs of non-mt and mt proteins, and the length for one of these gaps is indicated.…”

Section: Acknowledgmentsmentioning

confidence: 99%

Self‐splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family

Gorbalenya¹

1994

Protein Science

110

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Abstract:A new family of protein domains consisting of 50-80 amino acid residues is described. It is composed of nearly 40 members, including domains encoded by plastid and phage group I introns; mitochondrial, plastid, and bacterial group I1 introns; eubacterial genomes and plasmids; and phages. The name "EXIHH-HX,H" was coined for both domain and family. It is based on 2 most prominent amino acid sequence motifs, each encompassing a pair of highly conserved histidine residues in a specific arrangement: EXIHH and HX3H. The "His" motifs often alternate with amino-and carboxy-terminal motifs of a new type of Zn-finger-like structure CX2,4CX29. 54[CH]X2,3[CH].The EX,HH-HX3H domain in eubacterial E2-type bacteriocins and in phage RB3 (wild variant of phage T4) product of the nrdB group I intron was reported to be essential for DNA endonuclease activity of these proteins. In other proteins, the EXlHH-HX3H domain is hypothesized to possess DNase activity as well. Presumably, this activity promotes movement (rearrangement) of group I and group I1 introns encoding the EX,HH-HX3H domain and other gene targets. In the case of Escherichia coli restrictase McrA and possibly several related proteins, it appears to mediate the restriction of alien DNA molecules.Keywords: E2-type bacteriocins; mcrA restriction; nifD locus rearrangement; phage 9C31; phage T4 ORFs; zinc finger Self-splicing introns containing open reading frames (ORFs) are suggested to represent a sort of molecular commensalist that probably emerged relatively recently in the course of evolution. In the partnership, introns provide a convenient genetic system for maintaining and expression of protein-encoded sequences, and the products of ORFs assist introns in their splicing and/or promote intron movement in the genetic environment. The perfect and very puzzling correlation between the group of selfsplicing introns and the family of proteins "infecting" it has been

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The phage T4 nrdB intron: a deletion mutant of a version found in the wild.

Cited by 71 publications

References 52 publications

Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

Insertion of a homing endonuclease creates a genes-in-pieces ribonucleotide reductase that retains function

Phage display of intact domains at high copy number: A system based on SOC, the small outer capsid protein of bacteriophage T4

Self‐splicing group I and group II introns encode homologous (putative) DNA endonucleases of a new family

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