FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1969881177> ?p ?o ?g. }

• W1969881177 endingPage "29374" @default.
• W1969881177 startingPage "29367" @default.
• W1969881177 abstract "QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases. QDE-1 is an RNA- and DNA-dependent RNA polymerase that has functions in the RNA silencing and DNA repair pathways of the filamentous fungus Neurospora crassa. The crystal structure of the dimeric enzyme has been solved, and the fold of its catalytic core is related closely to that of eukaryotic DNA-dependent RNA polymerases. However, the specific activities of this multifunctional enzyme are still largely unknown. In this study, we characterized the in vitro activities of the N-terminally truncated QDE-1ΔN utilizing structure-based mutagenesis. Our results indicate that QDE-1 displays five distinct catalytic activities, which can be dissected by mutating critical amino acids or by altering reaction conditions. Our data also suggest that the RNA- and DNA-dependent activities have different modes for the initiation of RNA synthesis, which may reflect the mechanism that enables the polymerase to discriminate between template nucleic acids. Moreover, we show that QDE-1 is a highly potent terminal nucleotidyltransferase. Our results suggest that QDE-1 is able to regulate its activity mode depending on the template nucleic acid. This work extends our understanding of the biochemical properties of the QDE-1 enzyme and related RNA polymerases. Gene expression of most eukaryotic organisms is regulated by an immense assortment of small RNAs and proteins that associate with them. These various components form networks known as RNA silencing pathways, most important of which employ small interfering RNAs (siRNAs), microRNAs, or piwi-interacting RNAs to achieve sequence specificity (1Siomi H. Siomi M.C. Nature. 2009; 457: 396-404Crossref PubMed Scopus (518) Google Scholar, 2Moazed D. Nature. 2009; 457: 413-420Crossref PubMed Scopus (626) Google Scholar, 3Ghildiyal M. Zamore P.D. Nat. Rev. Genet. 2009; 10: 94-108Crossref PubMed Scopus (1845) Google Scholar). RNA silencing associated cell-encoded RNA-dependent RNA polymerases (RdRPs) 3The abbreviations used are: RdRPRNA-dependent RNA polymeraseDdRPDNA-dependent RNA polymerasedsdouble-strandedsssingle-strandedTNTaseterminal nucleotidyltransferasentnucleotide(s)qiRNAQDE-2-interacting small RNA. are found commonly as components of the RNA silencing pathways of plants, fungi, and nematodes (1Siomi H. Siomi M.C. Nature. 2009; 457: 396-404Crossref PubMed Scopus (518) Google Scholar, 4Rana T.M. Nat. Rev. Mol. Cell Biol. 2007; 8: 23-36Crossref PubMed Scopus (857) Google Scholar). The functions of cellular RdRPs have been largely elusive, but recent studies have shed some light on their enigmatic character. Caenorhabditis elegans RdRPs have been shown to synthesize secondary siRNAs that are important for amplifying the initial silencing signal (5Ruby J.G. Jan C. Player C. Axtell M.J. Lee W. Nusbaum C. Ge H. Bartel D.P. Cell. 2006; 127: 1193-1207Abstract Full Text Full Text PDF PubMed Scopus (745) Google Scholar, 6Pak J. Fire A. Science. 2007; 315: 241-244Crossref PubMed Scopus (451) Google Scholar, 7Sijen T. Steiner F.A. Thijssen K.L. Plasterk R.H. Science. 2007; 315: 244-247Crossref PubMed Scopus (314) Google Scholar). Tetrahymena termophila RdRP (Rdr1) is known to interact with Dicer to produce endogenous siRNAs, whereas the RdRP (Rdp1) of Schizosaccharomyces pombe is critical for heterochromatic gene silencing (2Moazed D. Nature. 2009; 457: 413-420Crossref PubMed Scopus (626) Google Scholar, 8Lee S.R. Collins K. Nat. Struct. Mol. Biol. 2007; 14: 604-610Crossref PubMed Scopus (78) Google Scholar, 9Sugiyama T. Cam H. Verdel A. Moazed D. Grewal S.I. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 152-157Crossref PubMed Scopus (228) Google Scholar). Arabidopsis thaliana has six genes that code for RdRPs, but only a few of these have been studied in detail (10Gazzani S. Lawrenson T. Woodward C. Headon D. Sablowski R. Science. 2004; 306: 1046-1048Crossref PubMed Scopus (258) Google Scholar, 11Curaba J. Chen X. J. Biol. Chem. 2008; 283: 3059-3066Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Most of the above studies imply that the main function of cellular RdRPs is in synthesizing siRNAs directly or making double-stranded RNA (dsRNA) from single-stranded RNA (ssRNA) templates to be used as Dicer substrate. For a long time, it was thought that cellular RdRPs are absent in insects and mammals, but recently, robust RdRP activities have been detected in Drosophila melanogaster and humans (12Lipardi C. Paterson B.M. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 15645-15650Crossref PubMed Scopus (56) Google Scholar, 13Maida Y. Yasukawa M. Furuuchi M. Lassmann T. Possemato R. Okamoto N. Kasim V. Hayashizaki Y. Hahn W.C. Masutomi K. Nature. 2009; 461: 230-235Crossref PubMed Scopus (303) Google Scholar) suggesting that cellular RdRPs may have crucial functions throughout the eukaryotic domain. RNA-dependent RNA polymerase DNA-dependent RNA polymerase double-stranded single-stranded terminal nucleotidyltransferase nucleotide(s) QDE-2-interacting small RNA. Neurospora crassa is a filamentous fungus that displays remarkable genomic stability (14Catalanotto C. Nolan T. Cogoni C. FEMS Microbiol. Lett. 2006; 254: 182-189Crossref PubMed Scopus (32) Google Scholar, 15Choudhary S. Lee H.C. Maiti M. He Q. Cheng P. Liu Q. Liu Y. Mol. Cell. Biol. 2007; 27: 3995-4005Crossref PubMed Scopus (54) Google Scholar). One of the cellular mechanisms that affect to this stability is an RNA-silencing pathway known as quelling (16Romano N. Macino G. Mol. Microbiol. 1992; 6: 3343-3353Crossref PubMed Scopus (576) Google Scholar). Quelling is initiated by repetitive genetic elements and is dependent on three genes: qde-1 (quelling defective) encoding an RdRP, qde-2 (a member of the Argonaute family), and qde-3 (a RecQ-like DNA helicase) (17Cogoni C. Macino G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 10233-10238Crossref PubMed Scopus (251) Google Scholar, 18Cogoni C. Macino G. Science. 1999; 286: 2342-2344Crossref PubMed Scopus (218) Google Scholar, 19Cogoni C. Macino G. Nature. 1999; 399: 166-169Crossref PubMed Scopus (515) Google Scholar). It has been shown that overexpression of QDE-1 results in increased silencing and that expression of hairpin dsRNA molecules abolishes the need of QDE-1 activity, suggesting that the primary function of QDE-1 is to synthesize dsRNA to be used as substrates for the two Dicers (DCL-1 and DCL-2) of Neurospora (15Choudhary S. Lee H.C. Maiti M. He Q. Cheng P. Liu Q. Liu Y. Mol. Cell. Biol. 2007; 27: 3995-4005Crossref PubMed Scopus (54) Google Scholar, 20Goldoni M. Azzalin G. Macino G. Cogoni C. Fungal Genet. Biol. 2004; 41: 1016-1024Crossref PubMed Scopus (90) Google Scholar, 21Catalanotto C. Azzalin G. Macino G. Cogoni C. Genes Dev. 2002; 16: 790-795Crossref PubMed Scopus (144) Google Scholar, 22Catalanotto C. Pallotta M. ReFalo P. Sachs M.S. Vayssie L. Macino G. Cogoni C. Mol. Cell. Biol. 2004; 24: 2536-2545Crossref PubMed Scopus (161) Google Scholar, 23Forrest E.C. Cogoni C. Macino G. Nucleic Acids Res. 2004; 32: 2123-2128Crossref PubMed Scopus (33) Google Scholar, 24Fulci V. Macino G. Curr. Opin. Microbiol. 2007; 10: 199-203Crossref PubMed Scopus (87) Google Scholar, 25Maiti M. Lee H.C. Liu Y. Genes Dev. 2007; 21: 590-600Crossref PubMed Scopus (98) Google Scholar). The Argonaute protein QDE-2 has slicer activity and interacts with an exonuclease known as QIP (25Maiti M. Lee H.C. Liu Y. Genes Dev. 2007; 21: 590-600Crossref PubMed Scopus (98) Google Scholar). The expression of QDE-2 is induced by dsRNA, and its steady-state levels are regulated by the DCLs (15Choudhary S. Lee H.C. Maiti M. He Q. Cheng P. Liu Q. Liu Y. Mol. Cell. Biol. 2007; 27: 3995-4005Crossref PubMed Scopus (54) Google Scholar). The biochemical roles of QDE-3 largely are unknown, but it has been suggested to have roles in both DNA repair and quelling (18Cogoni C. Macino G. Science. 1999; 286: 2342-2344Crossref PubMed Scopus (218) Google Scholar, 26Pickford A. Braccini L. Macino G. Cogoni C. Curr. Genet. 2003; 42: 220-227Crossref PubMed Scopus (22) Google Scholar, 27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar). The classical model of transgene quelling in Neurospora begins by RNA polymerase II and QDE-3 synthesizing an aberrant RNA molecule, which is recognized by QDE-1 and converted into dsRNA (24Fulci V. Macino G. Curr. Opin. Microbiol. 2007; 10: 199-203Crossref PubMed Scopus (87) Google Scholar). This dsRNA is digested to double-stranded siRNAs by the DCLs. These associate with QDE-2, which nicks the passenger strand of the siRNA that is degraded subsequently by the QIP exonuclease (25Maiti M. Lee H.C. Liu Y. Genes Dev. 2007; 21: 590-600Crossref PubMed Scopus (98) Google Scholar). QDE-2, now containing a single-stranded siRNA guide strand, finds its complementary mRNA (or aberrant RNA) targets, which leads to the silencing of both transgenic and endogenous transcripts. Recently, this model has been challenged by the discovery that quelling components have essential roles in the nucleus associated with DNA repair (27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar, 28Nolan T. Cecere G. Mancone C. Alonzi T. Tripodi M. Catalanotto C. Cogoni C. Nucleic Acids Res. 2008; 36: 532-538Crossref PubMed Scopus (26) Google Scholar). QDE-1 was shown to co-purify with ssDNA binding replication protein A (RPA), and DNA damage was shown to induce QDE-2 expression. Immunoprecipitation of QDE-2 from DNA damaged Neurospora cultures revealed a novel type of small RNAs known as QDE-2-interacting small RNAs that are mostly derived from the ribosomal DNA (rDNA) locus (27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar). qiRNA production is dependent on QDE-1, QDE-3, and the DCLs but not on QDE-2. Notably, QDE-2-interacting small RNAs are derived from aberrant RNAs that are synthesized by QDE-1 and not by any of the canonical RNA polymerases. QDE-1 was shown to have a robust DNA-dependent RNA polymerase (DdRP) activity, generating a DNA/RNA hybrid from an ssDNA template (27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar). Much insight into the structure and function of cellular RdRPs has come from the studies of a recombinant QDE-1 and its catalytically active C-terminal portion QDE-1ΔN (residues 377–1402 of the wild-type) (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 30Laurila M.R. Salgado P.S. Makeyev E.V. Nettelship J. Stuart D.I. Grimes J.M. Bamford D.H. J. Struct. Biol. 2005; 149: 111-115Crossref PubMed Scopus (19) Google Scholar, 31Salgado P.S. Koivunen M.R. Makeyev E.V. Bamford D.H. Stuart D.I. Grimes J.M. PLoS Biol. 2006; 4: e434Crossref PubMed Scopus (71) Google Scholar). The recombinant polymerase is able to initiate RNA synthesis without a primer and convert heterologous ssRNAs into double-stranded molecules. In addition to making full-length dsRNA copies of ssRNA templates, QDE-1 was observed to synthesize small 9–21-nt RNAs scattered along template RNAs (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The crystal structure of QDE-1ΔN showed that the molecule is a dimer and that the catalytic core has a fold that is related to those in eukaryotic DdRPs (31Salgado P.S. Koivunen M.R. Makeyev E.V. Bamford D.H. Stuart D.I. Grimes J.M. PLoS Biol. 2006; 4: e434Crossref PubMed Scopus (71) Google Scholar). In this study, we demonstrate that QDE-1ΔN displays five distinct in vitro activities. We use structure-based mutagenesis to show that the activities can be dissected by mutating critical amino acid residues and suggest that RdRP and DdRP activities have different initiation mechanisms and pH optima. The biochemical data presented in this study imply a recognition mechanism that discerns a DNA template from an RNA template. These results have broader ramifications in eukaryotic RNA- and DNA-dependent RNA polymerases associated with RNA silencing pathways. The expression vectors for QDE-1ΔN point mutants were generated by site-directed mutagenesis using PCR. The mutagenic primers are listed in supplemental Table S1. Plasmid pEM69 (30Laurila M.R. Salgado P.S. Makeyev E.V. Nettelship J. Stuart D.I. Grimes J.M. Bamford D.H. J. Struct. Biol. 2005; 149: 111-115Crossref PubMed Scopus (19) Google Scholar) encoding for a His-tagged QDE-1ΔN (missing amino acids 1–376) was used as a template in 50-μl PCR reactions each containing sense and antisense primers, and 2.5 units of PfuTurbo DNA polymerase (Stratagene). After completion, the reactions were treated with 10 units of DpnI (Fermentas) and transformed into CaCl2 competent Escherichia coli XL1-Blue cells (Stratagene). The correct constructs were verified by restriction enzyme analysis and sequencing. The recombinant proteins were expressed and purified as described previously for QDE-1ΔN (pEM69; (30Laurila M.R. Salgado P.S. Makeyev E.V. Nettelship J. Stuart D.I. Grimes J.M. Bamford D.H. J. Struct. Biol. 2005; 149: 111-115Crossref PubMed Scopus (19) Google Scholar)). Briefly, the plasmids were introduced into Saccharomyces cerevisiae strain INVSc1 (Invitrogen), the recombinant proteins were expressed at +28 °C for 22 h and purified to near homogeneity. The yeast cells were first harvested and disrupted by a French press. The cell lysates were then cleared by centrifugation, and the supernatants were loaded onto nickel-nitrilotriacetic acid affinity columns (Qiagen), washed with 5 mm and 25 mm imidazole-containing buffers, and eluted with 200 mm imidazole. Subsequently, the proteins were purified by HiTrapTM heparin HP and Q HP columns (GE Healthcare) and eluted by increasing NaCl gradients. The purified proteins were stored in 50 mm Tris-HCl (pH 8.0), 0.1 mm EDTA, 0.13% Triton X-100, 100 mm NaCl, and 62.5% glycerol at −20 °C. The oligomeric status of the recombinant polymerases was analyzed by size-exclusion chromatography using a Superdex 200 16/60 gel filtration column (GE Healthcare) with appropriate control proteins (Sigma). Plasmid pLM659 (32Gottlieb P. Strassman J. Qiao X. Frilander M. Frucht A. Mindich L. J. Virol. 1992; 66: 2611-2616Crossref PubMed Google Scholar) contains a cDNA copy of the S segment of bacteriophage ϕ6 under a T7 promoter. For the production of ssRNA, pLM659 was linearized by SmaI digestion, purified using a PCR purification kit (Qiagen), and used as a template for run-off transcription by T7 RNA polymerase. The template DNA was degraded with DNaseI (Promega) and the ssRNA purified by chloroform extraction and LiCl precipitation. To generate a ssDNA molecule of the same length and sequence, SmaI-digested pLM659 was used as a template in PCR reactions containing primers AO49 and AO50 (see supplemental Table S4) and Phusion® DNA polymerase (Finnzymes). AO50 contains a 5′-biotin. The biotinylated PCR product was immobilized onto Dynabeads® MyOneTM streptavidin C1 magnetic beads (Invitrogen) according to the manufacturer's instructions. The immobilized PCR product was dissolved by treating the DNA briefly with fresh 0.1 m NaOH. The ssDNA was precipitated with sodium acetate and ethanol and gel-purified through agarose gel electrophoresis. Prior to 5′-labeling, the ssRNA was treated with alkaline phosphatase (Finnzymes). Both ssRNA and ssDNA were 5′-labeled with [γ-32P]ATP (NEN Radiochemicals, PerkinElmer Life Sciences) and T4 polynucleotide kinase (Fermentas). M13mp18 ssDNA was purchased from New England Biolabs. The oligonuleotides (AO49–52) were purchased from biomers.net or Eurofins MWG Operon. Polymerase reactions were performed essentially as described (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 33Yang H. Makeyev E.V. Bamford D.H. J. Virol. 2001; 75: 11088-11095Crossref PubMed Scopus (38) Google Scholar). The standard QDE-1 reaction mixture contained 50 mm HEPES-KOH (pH 7.8), 20 mm ammonium acetate, 1 mm MgCl2, 1 mm MnCl2, 6% (w/v) polyethylene glycol 4000, 0.1 mm EDTA, 0.1% Triton X-100, 0.2 mm of each NTP, 1 unit/μl RNasin® ribonuclease inhibitor (Promega), and 0.01–0.02 μg/μl QDE-1ΔN. In some of the pH experiments, HEPES-KOH (pH 7.2–7.8) was replaced by Bis-Tris (pH 6.0–6.9) or Tris-HCl (pH 8.0–8.9). The ladder reactions were programmed with 5 mm MgCl2. Reactions were supplemented with 0.1 mCi/ml of [α-32P]UTP (GE Healthcare or NEN Radiochemicals, PerkinElmer Life Sciences) or other radioactive NTPs where indicated. The reactions were incubated at +30 °C for 1 h and quenched with U2 (8 m urea, 10 mm EDTA, 0.2% SDS, 6% (v/v) glycerol, 0.05% bromphenol blue, and 0.05% xylene cyanol) loading buffer. Some reaction products were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform:isoamyl alcohol (24:1), precipitated with NH4OAc and ethanol, and dissolved in milli-Q water. Some samples were treated with RNase T1 (Fermentas) for 15 min at +37 °C. The samples were subjected to standard Tris-Borate-EDTA or Tris-Acetate-EDTA agarose gel electrophoresis or denaturing, formaldehyde-containing agarose gel electrophoresis (34Sambrook J. Russell D. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York2001Google Scholar). The gels were visualized by ethidium bromide staining and dried, and radioactivity was detected by phosphorimaging (Fuji FLA-5000) and analyzed by densitometry with AIDA software (Raytest Isotopenmeβgeräte). Some samples were subjected to denaturing PAGE by urea-containing 20% sequencing gels. These were either desalted by Zeba Spin Desalting Columns (Thermo Scientific) or purified by phenol extraction and ethanol precipitation. Prior to loading, the samples were mixed with Gel Loading Buffer II (Ambion) and heated to +95 °C for 5 min. To elucidate the different catalytic activities of QDE-1ΔN, standard polymerization reactions were carried out with ssRNA or ssDNA templates in different conditions (Fig. 1A). Mixing QDE-1ΔN with ssRNA, all four NTPs and [α-32P]UTP resulted in dsRNA synthesis as well as decreased mobility (shifting) and labeling of the ssRNA template (lane 2). In addition, labeled RNA products were detected that varied in size from tens of nucleotides to several hundreds. Some of these products did not enter the agarose gel and remained in the wells. No labeled products were detected when the catalytically inactive QDE-1ΔNDA was used in the reaction mix (lane 3) (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). When the reactions were carried out with only UTP and trace amounts of [α-32P]UTP, the template ssRNA was efficiently labeled without dsRNA synthesis (lane 4), indicating that QDE-1ΔN is a potent terminal nucleotidyltransferase (TNTase). QDE-1ΔN displays also a strong DNA-dependent RNA polymerase activity (lane 6) (27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar). None of the template ssDNA migrates as template-sized but is very efficiently converted to the DNA/RNA hybrid form (lane 6). Again, substituting the ΔN polymerase with QDE-1ΔN DA abolishes this activity (lane 7). The TNTase activity also is very prominent with an ssDNA template (lane 8). Using only UTP as the substrate, the ssDNA template migrates at its normal position (upper panel) but is extensively labeled (lower panel). The above experiments show that QDE-1ΔN displays five distinct activities (Fig. 1, A and B): (i) RNA-dependent RNA polymerase activity (Fig. 1A, lane 2) (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), (ii) DNA-dependent RNA polymerase activity (Fig. 1A, lane 6) (27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar), (iii) ssRNA template shift and labeling activity (Fig. 1A, lane 2), (iv) TNTase activity (Fig. 1A, lanes 4 and 8), and (v) ladder activity (Fig. 1A, lanes 2, 6, and 9) (supplemental Fig. S1). Activities (i) and (ii) have been described previously (27Lee H.C. Chang S.S. Choudhary S. Aalto A.P. Maiti M. Bamford D.H. Liu Y. Nature. 2009; 459: 274-277Crossref PubMed Scopus (221) Google Scholar, 29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Activity (iii) has been suggested previously to result from the synthesis of 9–21-nt small RNAs that are scattered across the ssRNA template, as well as to be the main in vitro reaction product of QDE-1ΔN (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). However, in this work, the intensity of the ssRNA labeling was not significantly more extensive than the labeling of the dsRNA product (Fig. 1A, lane 2), suggesting that the “small RNAs” are not the main reaction product. The identity of this activity is further discussed below. TNTase activity (iv) (see below) has been detected previously in both viral and eukaryotic RdRPs, where nucleotides are added to the 3′-ends of the templates in a template-independent fashion (11Curaba J. Chen X. J. Biol. Chem. 2008; 283: 3059-3066Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 35Poranen M.M. Koivunen M.R. Bamford D.H. J. Virol. 2008; 82: 9254-9264Crossref PubMed Scopus (22) Google Scholar). Activity (v) (Fig. 1A and supplemental Fig. S1) was assigned as a ladder activity because it generates RNA products of all sizes. When the reaction products of a reaction without a template were analyzed on a denaturing sequencing gel, they migrated at one-nucleotide increments (starting from ∼8 nts) forming a “ladder” (supplemental Fig. S1A). This activity is template-independent because omitting the template does not affect the formation of the ladder (Fig. 1A, lane 9). However, the sensitivity of the ladder activity to varying reaction conditions suggests that it is an in vitro side reaction occurring at high enzyme and substrate conditions (supplemental Fig. S1). The in vitro activities of QDE-1ΔN are summarized schematically in Fig. 1B. As QDE-1 displays both RNA- and DNA-dependent RNA polymerase activities, we further studied the nature of these reactions. We programmed polymerization reactions with ssRNA or ssDNA templates of the same length and sequence, purified the reaction products, and analyzed them by denaturing formaldehyde-containing agarose gel electrophoresis. As controls we labeled the templates at the 5′-end with [γ-32P]ATP and polynucleotide kinase (Fig. 2A). As has been shown previously (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), most of the product that accumulated using an ssRNA template migrated more slowly than the template, indicating that the strands of the dsRNA molecule are covalently linked together (“back-priming”). In contrast, the products of the reaction using an ssDNA template migrated as template-sized or smaller, indicating that the mode of RNA synthesis initiation differs between these two templates. This result is further supported by an experiment where RdRP and DdRP reactions were carried out with an initiating nucleotide that was 32P-labeled at the γ-phosphate (Fig. 2B). In this experimental setup, the product RNA can be labeled only if the γ-phosphate remains within the first nucleotide of the new strand. Conversely, if RNA synthesis is initiated by back-priming, the γ-phosphate is removed from the product RNA. As expected, radioactivity was detected only in the double-stranded products of the ssDNA template (DNA/RNA hybrids) and the template-sized products of the ssRNA template (resulting from abortive initiation, see below). dsRNA was not labeled. In addition, we performed QDE-1ΔN activity assays with both ssRNA and ssDNA templates in the same reaction mixture simultaneously (supplemental Fig. S2). The RdRP or DdRP activities did not inhibit each other, but both templates were processed into products. The ssDNA template in this experiment was circular, indicating that QDE-1ΔN is able to initiate RNA synthesis without the need for a free 3′-end (supplemental Fig. S2). All in all, these data suggest that QDE-1 is able to discriminate between ssRNA and ssDNA templates. The crystal structure of QDE-1ΔN has been solved previously (31Salgado P.S. Koivunen M.R. Makeyev E.V. Bamford D.H. Stuart D.I. Grimes J.M. PLoS Biol. 2006; 4: e434Crossref PubMed Scopus (71) Google Scholar). Using the structural information, we designed eight point mutations that were predicted to functionally disrupt QDE-1ΔN (Fig. 3A and supplemental Fig. S3 and Tables S1 and S2). The constructs were transformed into S. cerevisiae, and the recombinant proteins were expressed. All of the mutant enzymes were soluble and purified to near homogeneity and behaved like the wild-type during purification (data not shown). Initial screening of the RdRP and DdRP activities of the point mutants revealed that they possessed characteristics that differed from the wild-type polymerase (supplemental Fig. S4A). As expected (as these were assumed to be catalytically essential aspartic acids), QDE-1ΔNDA (D1011A) (29Makeyev E.V. Bamford D.H. Mol. Cell. 2002; 10: 1417-1427Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and D1007A were catalytically completely inactive. Of the active point mutants, five (R738A, R944E, K1119W, M1357D, and M1357C) were chosen for more extensive studies due to their catalytic properties. Arg738 lies within a channel that is predicted to accommodate the reaction product of QDE-1ΔN and direct it away from the active site (Fig. 3A). The R944E mutation is predicted to partly block the communication tunnel that links the two active sites in a QDE-1ΔN dimer (Fig. 3A). The K1119W mutation was designed to occlude a pore in QDE-1ΔN that apparently allows substrate nucleotides to enter the active site. The M1357D and M1357C mutations are predicted to respectively weaken and lock together the dimeric interface of the QDE-1ΔN head domains (Fig. 3A). However, the interface of the entire QDE-1 dimer is so extensive that mutating Met1357 should not affect the oligomerization state of the enzyme. To confirm this, we performed analytical gel filtration chromatography with QDE-1ΔN WT in different conditions, some of the point mutant enzymes, and control proteins of various sizes (Table 1). Dimeric QDE-1ΔN is predicted to be ∼230 kDa in size, whereas a monomer would have the predicted size of ∼120 kDa (31Salgado P.S. Koivunen M.R. Makeyev E.V. Bamford D.H. Stuart D.I. Grimes J.M. PLoS Biol. 2006; 4: e434Crossref PubMed Scopus (71) Google Scholar). Our results establish that all QDE-1ΔN enzymes are dimeric, regardless of the surrounding pH (Table 1). This has been deduced previously from the crystal structure, as each of the subunits has >2000 Å2 of contact area with the neighboring subunit (31Salgado P.S. Koivunen M.R. Makeyev E.V. Bamford D.H. Stuart D.I. Grimes J.M. PLoS Biol. 2006; 4: e434Crossref PubMed Scopus (71) Google Scholar).TABLE 1Analytical gel filtrations of QDE-1ΔN mutant polymerasesProteinPeak elution timeminApoferritin (Sigma), 443 kDa113.47QDE-1ΔN WT129.52 (pH 6.3), 127.76 (pH 7.4), 128.03 (pH 8.9)QDE-1ΔN D1007A126.75QDE-1ΔN P964A127.92QDE-1ΔN R738A127.23QDE-1ΔN M1357D127.23QDE-1ΔN K1119W127.23QDE-1ΔN R944E127.12Alcohol dehydrogenase (Sigma), 150 kDa137.42 (pH 6.3), 137.31 (pH 7.4), 135.82 (pH 8.9) Open table in a new tab All of the five point mutants under closer scrutiny were catalytically active on both ssRNA and ssDNA templates (Fig. 3B). The catalytic activity of R738A is reduced to approximately half of that of the wild-type regardless of the template (Fig. 3C), in accordance with a nonspecific charge steering role for this residue. In addition, R738A is incapable of shifting and labeling the ssRNA template (Fig. 3B and supplemental Fig. S4A). The DdRP activity of R944E is close to that of the native polymerase. However, its RdRP activity is only ∼20% of the wild-type (Fig. 3C). It shifts the ssRNA template and labels it efficiently (Fig. 3B and supplemental Fi" @default.
• W1969881177 created "2016-06-24" @default.
• W1969881177 creator A5019055664 @default.
• W1969881177 creator A5022666879 @default.
• W1969881177 creator A5061540289 @default.
• W1969881177 creator A5071726265 @default.
• W1969881177 creator A5083982163 @default.
• W1969881177 date "2010-09-01" @default.
• W1969881177 modified "2023-09-29" @default.
• W1969881177 title "In Vitro Activities of the Multifunctional RNA Silencing Polymerase QDE-1 of Neurospora crassa*" @default.
• W1969881177 cites W1513811372 @default.
• W1969881177 cites W1522151190 @default.
• W1969881177 cites W1932753289 @default.
• W1969881177 cites W1970535781 @default.
• W1969881177 cites W1986640851 @default.
• W1969881177 cites W1988467918 @default.
• W1969881177 cites W1994307351 @default.
• W1969881177 cites W1994914565 @default.
• W1969881177 cites W2004271888 @default.
• W1969881177 cites W2016851067 @default.
• W1969881177 cites W2017733353 @default.
• W1969881177 cites W2029340312 @default.
• W1969881177 cites W2034206562 @default.
• W1969881177 cites W2037108634 @default.
• W1969881177 cites W2037141750 @default.
• W1969881177 cites W2058499120 @default.
• W1969881177 cites W2059635534 @default.
• W1969881177 cites W2063159592 @default.
• W1969881177 cites W2069099748 @default.
• W1969881177 cites W2073946012 @default.
• W1969881177 cites W2077353919 @default.
• W1969881177 cites W2077601414 @default.
• W1969881177 cites W2080356693 @default.
• W1969881177 cites W2084622414 @default.
• W1969881177 cites W2085742435 @default.
• W1969881177 cites W2102474285 @default.
• W1969881177 cites W2115225629 @default.
• W1969881177 cites W2119740058 @default.
• W1969881177 cites W2127043804 @default.
• W1969881177 cites W2128876450 @default.
• W1969881177 cites W2133313719 @default.
• W1969881177 cites W2138910750 @default.
• W1969881177 cites W2142762999 @default.
• W1969881177 cites W2151809557 @default.
• W1969881177 cites W2162674498 @default.
• W1969881177 doi "https://doi.org/10.1074/jbc.m110.139121" @default.
• W1969881177 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2937969" @default.
• W1969881177 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/20647305" @default.
• W1969881177 hasPublicationYear "2010" @default.
• W1969881177 type Work @default.
• W1969881177 sameAs 1969881177 @default.
• W1969881177 citedByCount "15" @default.
• W1969881177 countsByYear W19698811772012 @default.
• W1969881177 countsByYear W19698811772014 @default.
• W1969881177 countsByYear W19698811772015 @default.
• W1969881177 countsByYear W19698811772016 @default.
• W1969881177 countsByYear W19698811772018 @default.
• W1969881177 countsByYear W19698811772019 @default.
• W1969881177 countsByYear W19698811772020 @default.
• W1969881177 countsByYear W19698811772022 @default.
• W1969881177 crossrefType "journal-article" @default.
• W1969881177 hasAuthorship W1969881177A5019055664 @default.
• W1969881177 hasAuthorship W1969881177A5022666879 @default.
• W1969881177 hasAuthorship W1969881177A5061540289 @default.
• W1969881177 hasAuthorship W1969881177A5071726265 @default.
• W1969881177 hasAuthorship W1969881177A5083982163 @default.
• W1969881177 hasBestOaLocation W19698811771 @default.
• W1969881177 hasConcept C104317684 @default.
• W1969881177 hasConcept C119056186 @default.
• W1969881177 hasConcept C143065580 @default.
• W1969881177 hasConcept C166703698 @default.
• W1969881177 hasConcept C202751555 @default.
• W1969881177 hasConcept C2191507 @default.
• W1969881177 hasConcept C2776449523 @default.
• W1969881177 hasConcept C2776739539 @default.
• W1969881177 hasConcept C2776769606 @default.
• W1969881177 hasConcept C54355233 @default.
• W1969881177 hasConcept C67705224 @default.
• W1969881177 hasConcept C82381507 @default.
• W1969881177 hasConcept C86803240 @default.
• W1969881177 hasConcept C95444343 @default.
• W1969881177 hasConceptScore W1969881177C104317684 @default.
• W1969881177 hasConceptScore W1969881177C119056186 @default.
• W1969881177 hasConceptScore W1969881177C143065580 @default.
• W1969881177 hasConceptScore W1969881177C166703698 @default.
• W1969881177 hasConceptScore W1969881177C202751555 @default.
• W1969881177 hasConceptScore W1969881177C2191507 @default.
• W1969881177 hasConceptScore W1969881177C2776449523 @default.
• W1969881177 hasConceptScore W1969881177C2776739539 @default.
• W1969881177 hasConceptScore W1969881177C2776769606 @default.
• W1969881177 hasConceptScore W1969881177C54355233 @default.
• W1969881177 hasConceptScore W1969881177C67705224 @default.
• W1969881177 hasConceptScore W1969881177C82381507 @default.
• W1969881177 hasConceptScore W1969881177C86803240 @default.
• W1969881177 hasConceptScore W1969881177C95444343 @default.
• W1969881177 hasIssue "38" @default.
• W1969881177 hasLocation W19698811771 @default.
• W1969881177 hasLocation W19698811772 @default.

• next