Phage T4-coded Stp: Double-Edged Effector of Coupled DNA and tRNA-Restriction Systems

Penner, Michal; Morad, Ilan; Snyder, Lois; Kaufmann, Gabriel

doi:10.1006/jmbi.1995.0343

Cited by 75 publications

(62 citation statements)

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“…If M. smegmatis (or some other natural host for these 10 mycobacteriophages) does have an RNA restriction system, we would predict that only the mycobacteriophages with an RNA repair pathway are capable of inducing an RNA damage response. This idea is consistent with the findings that activation of the PrrC anticodon nuclease by T4 infection depends on a specific T4-encoded effector protein Stp (52).…”

Section: Discussionsupporting

confidence: 91%

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

Zhu

Yin

Shuman

2004

Journal of Biological Chemistry

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Coliphage T4 Pnkp is a bifunctional polynucleotide 5 -kinase/3 -phosphatase that catalyzes the end-healing steps of a RNA repair pathway. Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5 -kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3 -phosphatase module with an essential acyl-phosphatase motif, DX-DGT. Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/ kinase enzymes found in mammals and fission yeast. Whereas the phage 5 -kinases differ from each other in their preferences for phosphorylation of 5 overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase. We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response. Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo. A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.Breaks in the phosphodiester backbone of genomic DNA or essential RNA molecules can lead to cell death if not repaired. Such breaks can be triggered by physical or chemical insults, enzymatic hydrolysis, exposure to cytotoxic drugs, etc. Although DNA repair pathways have been the focus of much attention in light of the connections between DNA damage, mutagenesis, and cancer, there is an emerging appreciation that distinctive pathways exist to maintain or manipulate RNA structure in response to breakage events. Viruses provided the initial sources for the identification and characterization of enzymes that rectify DNA and RNA breaks (1-12) and viral model systems continue to illuminate the mechanisms, atomic structures, and biological roles of such enzymes (13-17).When breakage of a 3Ј-5Ј phosphodiester results in the formation of 5Ј-PO 4 and 3Ј-OH termini at the break site, the ends can be rejoined to each other (or to novel partner strands) by the action of DNA-specific or RNA-specific polynucleotide ligases. However, when breakage occurs with the opposite polarity, resulting in 5Ј-OH and 3Ј-PO 4 (or 2Ј,3Ј-cyclic PO 4 ) termini, the broken ends must be "healed" before they can be sealed. Healing entails two steps: (i) hydrolysis of the 3Ј-PO 4 (or 2Ј,3Ј-cyclic phosphate) to form a 3Ј-OH and (ii) phosphorylation of the 5Ј-OH to form a 5Ј-PO 4 end.The bacteriophage T4 proteins polynucleotide kinase/phosphatase (Pnkp) and RNA ligase 1 (Rnl1) are the prototypal RNA healing and sealing enzymes (1-3, 8 -1...

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Section: Discussionsupporting

confidence: 91%

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

Zhu

Yin

Shuman

2004

Journal of Biological Chemistry

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“…It comprises the core ACNase polypeptide PrrC and type IC DNA restriction-modification enzyme EcoprrI that masks PrrCs activity (2)(3)(4)(5). The phage T4-coded peptide Stp inhibits EcoprrI DNA restriction and activates ACNase (6). Cleavage of tRNA Lys 5Ј to the wobble base ensues, yielding 2Ј:3Ј-cyclic phosphate (2Ј:3Ј-pϾ) and 5Ј-OH termini.…”

Section: A Trnamentioning

confidence: 99%

“…: 972-3-640-9067; Fax: 972-3-640-6834; E-mail: gabika@tauex.tau.ac.il. 1 The abbreviations used are: ACNase, anticodon nuclease; ASL, anticodon stem and loop; t 6 A, threonylcarbamoyl adenosine; mnm 5 s 2 U, 5-methylaminomethyl-2-thiouridine; mcm 5 s 2 U, 5-methoxycarbonyl methyl-2-thiouridine.…”

Section: A Trnamentioning

confidence: 99%

Structural Features of tRNALys Favored by Anticodon Nuclease as Inferred from Reactivities of Anticodon Stem and Loop Substrate Analogs

Jiang¹,

Blanga²,

Amitsur³

et al. 2002

Journal of Biological Chemistry

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The bacterial tRNA Lys -specific PrrC-anticodon nuclease efficiently cleaved an anticodon stem-loop (ASL) oligoribonucleotide containing the natural modified bases, suggesting this region harbors the specificity determinants. Assays of ASL analogs indicated that the 6-threonylcarbamoyl adenosine modification (t 6 A37) enhances the reactivity. The side chain of the modified wobble base 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U34) has a weaker positive effect depending on the context of other modifications. The s 2 U34 modification apparently has none and the pseudouridine (⌿39) was inhibitory in most modification contexts. GC-rich but not IC-rich stems abolished the activity. Correlating the reported structural effects of the base modifications with their effects on anticodon nuclease activity suggests preference for substrates where the anticodon nucleotides assume a stacked A-RNA conformation and base pairing interactions in the stem are destabilized. Moreover, the proposal that PrrC residue Asp 287 contacts mnm 5 s 2 U34 was reinforced by the observations that the mammalian tRNA Lys-3 wobble base 5-methoxycarbonyl methyl-2-thiouridine (mcm 5 s 2 U) is inhibitory and that the D287H mutant favors tRNA Lys-3 over Escherichia coli tRNA Lys . The detection of this mutation and ability of PrrC to cleave the isolated ASL suggest that anticodon nuclease may be used to cleave tRNA Lys-3 primer molecules annealed to the genomic RNA template of the human immunodeficiency virus.

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“…In Escherichia coli strains containing the prr locus, the host cell tRNA Lys is cleaved 5Ј to the wobble position by a T4-induced anticodon nuclease. If Pnk and RNA ligase are not present, the synthesis of viral proteins is blocked by depletion of tRNA Lys , and the phage cannot replicate (2,(12)(13)(14). This pathway represents an RNA-based restriction system of host defense against a foreign invader.…”

mentioning

confidence: 99%

Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains

Shuman²

2002

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

150

174

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RNA ligases participate in repair, splicing, and editing pathways that either reseal broken RNAs or alter their primary structure. Bacteriophage T4 RNA ligase (gp63) is the best-studied member of this class of enzymes, which includes yeast tRNA ligase and trypanosome RNA-editing ligases. Here, we identified another RNA ligase from the bacterial domain-a second RNA ligase (Rnl2) encoded by phage T4. Purified Rnl2 (gp24.1) catalyzes intramolecular and intermolecular RNA strand joining through ligase-adenylate and RNA-adenylate intermediates. Mutational analysis identifies amino acids required for the ligase-adenylation or phosphodiester synthesis steps of the ligation reaction. The catalytic residues of Rnl2 are located within nucleotidyl transferase motifs I, IV, and V that are conserved in DNA ligases and RNA capping enzymes. Rnl2 has scant amino acid similarity to T4 gp63. Rather, Rnl2 exemplifies a distinct ligase family, defined by variant motifs, that includes the trypanosome-editing ligases and a group of putative RNA ligases encoded by eukaryotic viruses (baculoviruses and an entomopoxvirus) and many species of archaea. These findings have implications for the evolution of covalent nucleotidyl transferases and virus-host dynamics based on RNA restriction and repair.T 4 RNA ligase (1) is the founding member of a family of RNA end-joining enzymes involved in RNA repair, splicing, and editing pathways (2-8). T4 RNA ligase joins 3Ј OH and 5Ј PO 4 RNA termini by means of three nucleotidyl transfer steps similar to those of DNA ligases (9-11).Step 1 is the reaction of RNA ligase with ATP to form a covalent ligase-(lysyl-N)-AMP intermediate. In step 2, the AMP is transferred to a 5Ј PO 4 RNA end to form an RNA-adenylate intermediate (AppRNA). In step 3, attack by an RNA 3Ј OH on RNA-adenylate seals the ends and releases AMP. T4 RNA ligase can join two single-stranded RNA molecules without a complementary bridging polynucleotide. T4 RNA ligase can also catalyze intramolecular ligation to form a covalently closed RNA circle. RNA ligase has been a powerful tool in the synthesis of RNAs of defined sequence, RNA 3Ј end modification, RNA 3Ј end-labeling, RNA sequencing, and structural analysis (11).During T4 infection in vivo, the RNA ligase, together with T4 polynucleotide kinase (Pnk), performs an RNA repair function that remodels and then seals broken tRNA ends. In Escherichia coli strains containing the prr locus, the host cell tRNA Lys is cleaved 5Ј to the wobble position by a T4-induced anticodon nuclease. If Pnk and RNA ligase are not present, the synthesis of viral proteins is blocked by depletion of tRNA Lys , and the phage cannot replicate (2,(12)(13)(14). This pathway represents an RNA-based restriction system of host defense against a foreign invader. tRNA anticodon nuclease systems are present in other pathogenic bacteria (14). The bacterial toxins colicins D and E5 are also anticodon nucleases that attack specific tRNAs (15). Thus, RNA repair enzymology has broad significance as a means to combat, or reco...

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Phage T4-coded Stp: Double-Edged Effector of Coupled DNA and tRNA-Restriction Systems

Cited by 75 publications

References 0 publications

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

Characterization of Polynucleotide Kinase/Phosphatase Enzymes from Mycobacteriophages Omega and Cjw1 and Vibriophage KVP40

Structural Features of tRNALys Favored by Anticodon Nuclease as Inferred from Reactivities of Anticodon Stem and Loop Substrate Analogs

Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains

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