메뉴 건너뛰기




Volumn 111, Issue 32, 2014, Pages

Single-molecule FRET reveals a corkscrew RNA structure for the polymerase-bound influenza virus promoter

Author keywords

[No Author keywords available]

Indexed keywords

FLUORESCENT DYE; RECOMBINANT ENZYME; RNA DIRECTED RNA POLYMERASE; SINGLE STRANDED RNA; VIRUS RNA;

EID: 84905974849     PISSN: 00278424     EISSN: 10916490     Source Type: Journal    
DOI: 10.1073/pnas.1406056111     Document Type: Article
Times cited : (47)

References (51)
  • 1
    • 84863754680 scopus 로고    scopus 로고
    • An overlapping protein-coding region in influenza A virus segment 3 modulates the host response
    • Jagger BW, et al. (2012) An overlapping protein-coding region in influenza A virus segment 3 modulates the host response. Science 337(6091):199-204
    • (2012) Science , vol.337 , Issue.6091 , pp. 199-204
    • Jagger, B.W.1
  • 2
    • 84870834635 scopus 로고    scopus 로고
    • Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain
    • Wise HM, et al. (2012) Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain. PLoS Pathog 8(11): e1002998
    • (2012) PLoS Pathog , vol.8 , Issue.11
    • Wise, H.M.1
  • 3
    • 67749133883 scopus 로고    scopus 로고
    • A complicated message: Identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA
    • Wise HM, et al. (2009) A complicated message: Identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA. J Virol 83(16):8021-8031
    • (2009) J Virol , vol.83 , Issue.16 , pp. 8021-8031
    • Wise, H.M.1
  • 4
    • 84874676641 scopus 로고    scopus 로고
    • Identification of novel influenza A virus proteins translated from PA mRNA
    • Muramoto Y, Noda T, Kawakami E, Akkina R, Kawaoka Y (2013) Identification of novel influenza A virus proteins translated from PA mRNA. J Virol 87(5):2455-2462
    • (2013) J Virol , vol.87 , Issue.5 , pp. 2455-2462
    • Muramoto, Y.1    Noda, T.2    Kawakami, E.3    Akkina, R.4    Kawaoka, Y.5
  • 5
    • 0018848920 scopus 로고
    • The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity
    • Desselberger U, Racaniello VR, Zazra JJ, Palese P (1980) The 3' and 5'-terminal sequences of influenza A, B and C virus RNA segments are highly conserved and show partial inverted complementarity. Gene 8(3):315-328
    • (1980) Gene , vol.8 , Issue.3 , pp. 315-328
    • Desselberger, U.1    Racaniello, V.R.2    Zazra, J.J.3    Palese, P.4
  • 6
    • 0018674114 scopus 로고
    • 5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus
    • Robertson JS (1979) 5' and 3' terminal nucleotide sequences of the RNA genome segments of influenza virus. Nucleic Acids Res 6(12):3745-3757
    • (1979) Nucleic Acids Res , vol.6 , Issue.12 , pp. 3745-3757
    • Robertson, J.S.1
  • 7
    • 0345742563 scopus 로고    scopus 로고
    • Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'-and 5'-terminal sequences
    • Barr JN, Wertz GW (2004) Bunyamwera bunyavirus RNA synthesis requires cooperation of 3'-and 5'-terminal sequences. J Virol 78(3):1129-1138
    • (2004) J Virol , vol.78 , Issue.3 , pp. 1129-1138
    • Barr, J.N.1    Wertz, G.W.2
  • 8
    • 59749084360 scopus 로고    scopus 로고
    • Genome cyclization as strategy for flavivirus RNA replication
    • Villordo SM, Gamarnik AV (2009) Genome cyclization as strategy for flavivirus RNA replication. Virus Res 139(2):230-239
    • (2009) Virus Res , vol.139 , Issue.2 , pp. 230-239
    • Villordo, S.M.1    Gamarnik, A.V.2
  • 9
    • 84880367286 scopus 로고    scopus 로고
    • The RNA polymerase of influenza a virus: Mechanisms of viral transcription and replication
    • Fodor E (2013) The RNA polymerase of influenza a virus: Mechanisms of viral transcription and replication. Acta Virol 57(2):113-122
    • (2013) Acta Virol , vol.57 , Issue.2 , pp. 113-122
    • Fodor, E.1
  • 10
    • 78651452290 scopus 로고    scopus 로고
    • Suppression of avian influenza transmission in genetically modified chickens
    • Lyall J, et al. (2011) Suppression of avian influenza transmission in genetically modified chickens. Science 331(6014):223-226
    • (2011) Science , vol.331 , Issue.6014 , pp. 223-226
    • Lyall, J.1
  • 11
    • 0028123774 scopus 로고
    • Recombinant influenza virus polymerase: Requirement of both 5' and 3' viral ends for endonuclease activity
    • Hagen M, Chung TD, Butcher JA, Krystal M (1994) Recombinant influenza virus polymerase: Requirement of both 5' and 3' viral ends for endonuclease activity. J Virol 68(3):1509-1515
    • (1994) J Virol , vol.68 , Issue.3 , pp. 1509-1515
    • Hagen, M.1    Chung, T.D.2    Butcher, J.A.3    Krystal, M.4
  • 12
    • 0029048936 scopus 로고
    • Differential activation of the influenza virus polymerase via template RNA binding
    • Cianci C, Tiley L, Krystal M (1995) Differential activation of the influenza virus polymerase via template RNA binding. J Virol 69(7):3995-3999
    • (1995) J Virol , vol.69 , Issue.7 , pp. 3995-3999
    • Cianci, C.1    Tiley, L.2    Krystal, M.3
  • 13
    • 0032889312 scopus 로고    scopus 로고
    • Characterization of influenza virus PB1 protein binding to viral RNA: Two separate regions of the protein contribute to the interaction domain
    • González S, Ortín J (1999) Characterization of influenza virus PB1 protein binding to viral RNA: Two separate regions of the protein contribute to the interaction domain. J Virol 73(1):631-637
    • (1999) J Virol , vol.73 , Issue.1 , pp. 631-637
    • González, S.1    Ortín, J.2
  • 14
    • 0033169059 scopus 로고    scopus 로고
    • Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates
    • González S, Ortín J (1999) Distinct regions of influenza virus PB1 polymerase subunit recognize vRNA and cRNA templates. EMBO J 18(13):3767-3775
    • (1999) EMBO J , vol.18 , Issue.13 , pp. 3767-3775
    • González, S.1    Ortín, J.2
  • 15
    • 0032189818 scopus 로고    scopus 로고
    • RNA-dependent activation of primer RNA production by influenza virus polymerase: Different regions of the same protein subunit constitute the two required RNA-binding sites
    • Li ML, Ramirez BC, Krug RM (1998) RNA-dependent activation of primer RNA production by influenza virus polymerase: Different regions of the same protein subunit constitute the two required RNA-binding sites. EMBO J 17(19):5844-5852
    • (1998) EMBO J , vol.17 , Issue.19 , pp. 5844-5852
    • Li, M.L.1    Ramirez, B.C.2    Krug, R.M.3
  • 16
    • 33644598441 scopus 로고    scopus 로고
    • A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase
    • Jung TE, Brownlee GG (2006) A new promoter-binding site in the PB1 subunit of the influenza A virus polymerase. J Gen Virol 87(Pt 3):679-688
    • (2006) J Gen Virol , vol.87 , Issue.PART 3 , pp. 679-688
    • Jung, T.E.1    Brownlee, G.G.2
  • 17
    • 0028228459 scopus 로고
    • Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs
    • Tiley LS, Hagen M, Matthews JT, Krystal M (1994) Sequence-specific binding of the influenza virus RNA polymerase to sequences located at the 5' ends of the viral RNAs. J Virol 68(8):5108-5116
    • (1994) J Virol , vol.68 , Issue.8 , pp. 5108-5116
    • Tiley, L.S.1    Hagen, M.2    Matthews, J.T.3    Krystal, M.4
  • 18
    • 0037443870 scopus 로고    scopus 로고
    • Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA
    • Lee MT, Klumpp K, Digard P, Tiley L (2003) Activation of influenza virus RNA polymerase by the 5' and 3' terminal duplex of genomic RNA. Nucleic Acids Res 31(6): 1624-1632
    • (2003) Nucleic Acids Res , vol.31 , Issue.6 , pp. 1624-1632
    • Lee, M.T.1    Klumpp, K.2    Digard, P.3    Tiley, L.4
  • 19
    • 0035845528 scopus 로고    scopus 로고
    • Structural features of an influenza virus promoter and their implications for viral RNA synthesis
    • Bae SH, et al. (2001) Structural features of an influenza virus promoter and their implications for viral RNA synthesis. Proc Natl Acad Sci USA 98(19):10602-10607
    • (2001) Proc Natl Acad Sci USA , vol.98 , Issue.19 , pp. 10602-10607
    • Bae, S.H.1
  • 20
    • 0033105857 scopus 로고    scopus 로고
    • Structure of influenza virus panhandle RNA studied by NMR spectroscopy and molecular modeling
    • Cheong HK, Cheong C, Lee YS, Seong BL, Choi BS (1999) Structure of influenza virus panhandle RNA studied by NMR spectroscopy and molecular modeling. Nucleic Acids Res 27(5):1392-1397
    • (1999) Nucleic Acids Res , vol.27 , Issue.5 , pp. 1392-1397
    • Cheong, H.K.1    Cheong, C.2    Lee, Y.S.3    Seong, B.L.4    Choi, B.S.5
  • 21
    • 0029853503 scopus 로고    scopus 로고
    • Promoter elements in the influenza vRNA terminal structure
    • Flick R, Neumann G, Hoffmann E, Neumeier E, Hobom G (1996) Promoter elements in the influenza vRNA terminal structure. RNA 2(10):1046-1057
    • (1996) RNA , vol.2 , Issue.10 , pp. 1046-1057
    • Flick, R.1    Neumann, G.2    Hoffmann, E.3    Neumeier, E.4    Hobom, G.5
  • 22
    • 0034959821 scopus 로고    scopus 로고
    • Hairpin loop structure in the 3' arm of the influenza A virus virion RNA promoter is required for endonuclease activity
    • Leahy MB, Dobbyn HC, Brownlee GG (2001) Hairpin loop structure in the 3' arm of the influenza A virus virion RNA promoter is required for endonuclease activity. J Virol 75(15):7042-7049
    • (2001) J Virol , vol.75 , Issue.15 , pp. 7042-7049
    • Leahy, M.B.1    Dobbyn, H.C.2    Brownlee, G.G.3
  • 23
    • 0034749284 scopus 로고    scopus 로고
    • Mutagenic analysis of the 5' arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity
    • Leahy MB, Pritlove DC, Poon LL, Brownlee GG (2001) Mutagenic analysis of the 5' arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity. J Virol 75(1):134-142
    • (2001) J Virol , vol.75 , Issue.1 , pp. 134-142
    • Leahy, M.B.1    Pritlove, D.C.2    Poon, L.L.3    Brownlee, G.G.4
  • 24
    • 0032980704 scopus 로고    scopus 로고
    • A hairpin loop at the 5' end of influenza A virus virion RNA is required for synthesis of poly(A)+ mRNA in vitro
    • Pritlove DC, Poon LL, Devenish LJ, Leahy MB, Brownlee GG (1999) A hairpin loop at the 5' end of influenza A virus virion RNA is required for synthesis of poly(A)+ mRNA in vitro. J Virol 73(3):2109-2114
    • (1999) J Virol , vol.73 , Issue.3 , pp. 2109-2114
    • Pritlove, D.C.1    Poon, L.L.2    Devenish, L.J.3    Leahy, M.B.4    Brownlee, G.G.5
  • 25
    • 0036635342 scopus 로고    scopus 로고
    • The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter
    • Brownlee GG, Sharps JL (2002) The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter. J Virol 76(14):7103-7113
    • (2002) J Virol , vol.76 , Issue.14 , pp. 7103-7113
    • Brownlee, G.G.1    Sharps, J.L.2
  • 26
    • 2942650138 scopus 로고    scopus 로고
    • Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules
    • Kapanidis AN, et al. (2004) Fluorescence-aided molecule sorting: Analysis of structure and interactions by alternating-laser excitation of single molecules. Proc Natl Acad Sci USA 101(24):8936-8941
    • (2004) Proc Natl Acad Sci USA , vol.101 , Issue.24 , pp. 8936-8941
    • Kapanidis, A.N.1
  • 27
    • 84880318919 scopus 로고    scopus 로고
    • Conformational landscapes of DNA polymerase i and mutator derivatives establish fidelity checkpoints for nucleotide insertion
    • Hohlbein J, et al. (2013) Conformational landscapes of DNA polymerase I and mutator derivatives establish fidelity checkpoints for nucleotide insertion. Nat Commun 4:2131
    • (2013) Nat Commun , vol.4 , pp. 2131
    • Hohlbein, J.1
  • 28
    • 84880337255 scopus 로고    scopus 로고
    • Rotavirus mRNAS are released by transcript-specific channels in the double-layered viral capsid
    • Periz J, et al. (2013) Rotavirus mRNAS are released by transcript-specific channels in the double-layered viral capsid. Proc Natl Acad Sci USA 110(29):12042-12047
    • (2013) Proc Natl Acad Sci USA , vol.110 , Issue.29 , pp. 12042-12047
    • Periz, J.1
  • 29
    • 84887265188 scopus 로고    scopus 로고
    • Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus
    • York A, Hengrung N, Vreede FT, Huiskonen JT, Fodor E (2013) Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus. Proc Natl Acad Sci USA 110(45):E4238-E4245
    • (2013) Proc Natl Acad Sci USA , vol.110 , Issue.45
    • York, A.1    Hengrung, N.2    Vreede, F.T.3    Huiskonen, J.T.4    Fodor, E.5
  • 30
    • 0027240154 scopus 로고
    • Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter
    • Fodor E, Seong BL, Brownlee GG (1993) Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter. J Gen Virol 74(Pt 7):1327-1333
    • (1993) J Gen Virol , vol.74 , Issue.PART 7 , pp. 1327-1333
    • Fodor, E.1    Seong, B.L.2    Brownlee, G.G.3
  • 31
    • 0028245360 scopus 로고
    • The influenza virus panhandle is involved in the initiation of transcription
    • Fodor E, Pritlove DC, Brownlee GG (1994) The influenza virus panhandle is involved in the initiation of transcription. J Virol 68(6):4092-4096
    • (1994) J Virol , vol.68 , Issue.6 , pp. 4092-4096
    • Fodor, E.1    Pritlove, D.C.2    Brownlee, G.G.3
  • 32
  • 33
    • 78049249486 scopus 로고    scopus 로고
    • Sensing DNA opening in transcription using quenchable Förster resonance energy transfer
    • Cordes T, et al. (2010) Sensing DNA opening in transcription using quenchable Förster resonance energy transfer. Biochemistry 49(43):9171-9180
    • (2010) Biochemistry , vol.49 , Issue.43 , pp. 9171-9180
    • Cordes, T.1
  • 34
    • 22144492905 scopus 로고    scopus 로고
    • Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation
    • Lee NK, et al. (2005) Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys J 88(4):2939-2953
    • (2005) Biophys J , vol.88 , Issue.4 , pp. 2939-2953
    • Lee, N.K.1
  • 35
    • 84893362271 scopus 로고    scopus 로고
    • Alternating-laser excitation: Single-molecule FRET and beyond
    • Hohlbein J, Craggs TD, Cordes T (2014) Alternating-laser excitation: Single-molecule FRET and beyond. Chem Soc Rev 43(4):1156-1171
    • (2014) Chem Soc Rev , vol.43 , Issue.4 , pp. 1156-1171
    • Hohlbein, J.1    Craggs, T.D.2    Cordes, T.3
  • 36
    • 84870852782 scopus 로고    scopus 로고
    • A toolkit and benchmark study for FRET-restrained high-precision structural modeling
    • Kalinin S, et al. (2012) A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat Methods 9(12):1218-1225
    • (2012) Nat Methods , vol.9 , Issue.12 , pp. 1218-1225
    • Kalinin, S.1
  • 37
    • 84870807256 scopus 로고    scopus 로고
    • Six steps closer to FRET-driven structural biology
    • Craggs TD, Kapanidis AN (2012) Six steps closer to FRET-driven structural biology. Nat Methods 9(12):1157-1158
    • (2012) Nat Methods , vol.9 , Issue.12 , pp. 1157-1158
    • Craggs, T.D.1    Kapanidis, A.N.2
  • 38
    • 67249130012 scopus 로고    scopus 로고
    • The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit
    • Dias A, et al. (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458(7240):914-918
    • (2009) Nature , vol.458 , Issue.7240 , pp. 914-918
    • Dias, A.1
  • 39
    • 67249100913 scopus 로고    scopus 로고
    • Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site
    • Yuan P, et al. (2009) Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site. Nature 458(7240):909-913
    • (2009) Nature , vol.458 , Issue.7240 , pp. 909-913
    • Yuan, P.1
  • 40
    • 67249108203 scopus 로고    scopus 로고
    • Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication
    • Jorba N, Coloma R, Ortín J (2009) Genetic trans-complementation establishes a new model for influenza virus RNA transcription and replication. PLoS Pathog 5(5): e1000462
    • (2009) PLoS Pathog , vol.5 , Issue.5
    • Jorba, N.1    Coloma, R.2    Ortín, J.3
  • 41
    • 77954912141 scopus 로고    scopus 로고
    • Influenza A virus-generated small RNAs regulate the switch from transcription to replication
    • Perez JT, et al. (2010) Influenza A virus-generated small RNAs regulate the switch from transcription to replication. Proc Natl Acad Sci USA 107(25):11525-11530
    • (2010) Proc Natl Acad Sci USA , vol.107 , Issue.25 , pp. 11525-11530
    • Perez, J.T.1
  • 42
    • 79959544394 scopus 로고    scopus 로고
    • Biophysical analysis of influenza A virus RNA promoter at physiological temperatures
    • Noble E, et al. (2011) Biophysical analysis of influenza A virus RNA promoter at physiological temperatures. J Biol Chem 286(26):22965-22970
    • (2011) J Biol Chem , vol.286 , Issue.26 , pp. 22965-22970
    • Noble, E.1
  • 43
    • 84896729143 scopus 로고    scopus 로고
    • Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates
    • Craggs TD, Hutton RD, Brenlla A, White MF, Penedo JC (2014) Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates. Nucleic Acids Res 42(3):1857-1872
    • (2014) Nucleic Acids Res , vol.42 , Issue.3 , pp. 1857-1872
    • Craggs, T.D.1    Hutton, R.D.2    Brenlla, A.3    White, M.F.4    Penedo, J.C.5
  • 44
    • 0036720769 scopus 로고    scopus 로고
    • A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs
    • Fodor E, et al. (2002) A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J Virol 76(18):8989-9001
    • (2002) J Virol , vol.76 , Issue.18 , pp. 8989-9001
    • Fodor, E.1
  • 45
    • 76249127905 scopus 로고    scopus 로고
    • Conformational transitions in DNA polymerase i revealed by single-molecule FRET
    • Santoso Y, et al. (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. Proc Natl Acad Sci USA 107(2):715-720
    • (2010) Proc Natl Acad Sci USA , vol.107 , Issue.2 , pp. 715-720
    • Santoso, Y.1
  • 46
    • 77954785228 scopus 로고    scopus 로고
    • Characterizing single-molecule FRET dynamics with probability distribution analysis
    • Santoso Y, Torella JP, Kapanidis AN (2010) Characterizing single-molecule FRET dynamics with probability distribution analysis. ChemPhysChem 11(10):2209-2219
    • (2010) ChemPhysChem , vol.11 , Issue.10 , pp. 2209-2219
    • Santoso, Y.1    Torella, J.P.2    Kapanidis, A.N.3
  • 47
    • 84874113188 scopus 로고    scopus 로고
    • The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: Implications for transcription start-site selection
    • Robb NC, et al. (2013) The transcription bubble of the RNA polymerase-promoter open complex exhibits conformational heterogeneity and millisecond-scale dynamics: Implications for transcription start-site selection. J Mol Biol 425(5):875-885
    • (2013) J Mol Biol , vol.425 , Issue.5 , pp. 875-885
    • Robb, N.C.1
  • 48
    • 23744455796 scopus 로고    scopus 로고
    • Alternating-laser excitation of single molecules
    • Kapanidis AN, et al. (2005) Alternating-laser excitation of single molecules. Acc Chem Res 38(7):523-533
    • (2005) Acc Chem Res , vol.38 , Issue.7 , pp. 523-533
    • Kapanidis, A.N.1
  • 49
    • 77949447172 scopus 로고    scopus 로고
    • RNAstructure: Software for RNA secondary structure prediction and analysis
    • Reuter JS, Mathews DH (2010) RNAstructure: Software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11:129
    • (2010) BMC Bioinformatics , vol.11 , pp. 129
    • Reuter, J.S.1    Mathews, D.H.2
  • 50
    • 77955405123 scopus 로고    scopus 로고
    • Assemble: An interactive graphical tool to analyze and build RNA architectures at the 2D and 3D levels
    • Jossinet F, Ludwig TE, Westhof E (2010) Assemble: An interactive graphical tool to analyze and build RNA architectures at the 2D and 3D levels. Bioinformatics 26(16): 2057-2059
    • (2010) Bioinformatics , vol.26 , Issue.16 , pp. 2057-2059
    • Jossinet, F.1    Ludwig, T.E.2    Westhof, E.3
  • 51
    • 80054682639 scopus 로고    scopus 로고
    • Application of the nano-positioning system to the analysis of fluorescence resonance energy transfer networks
    • Muschielok A, Michaelis J (2011) Application of the nano-positioning system to the analysis of fluorescence resonance energy transfer networks. J Phys Chem B 115(41): 11927-11937.
    • (2011) J Phys Chem B , vol.115 , Issue.41 , pp. 11927-11937
    • Muschielok, A.1    Michaelis, J.2


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.