메뉴 건너뛰기




Volumn 10, Issue 11, 2004, Pages 1730-1739

A base triple in the Tetrahymena group I core affects the reaction equilibrium via a threshold effect

Author keywords

Base triple; Catalysis; Conformational changes; Group I intron; RNA structure; Threshold effects

Indexed keywords

L 21 SCAI RIBOZYME; RIBOZYME; UNCLASSIFIED DRUG;

EID: 7044220277     PISSN: 13558382     EISSN: None     Source Type: Journal    
DOI: 10.1261/rna.7118104     Document Type: Article
Times cited : (9)

References (50)
  • 1
    • 3042848954 scopus 로고    scopus 로고
    • Crystal structure of a self-splicing group I intron with both exons
    • Adams, P.L., Stahley, M.R., Kosek, A.B., Wang, J., and Strobel, S.A. 2004. Crystal structure of a self-splicing group I intron with both exons. Nature 430: 45-50.
    • (2004) Nature , vol.430 , pp. 45-50
    • Adams, P.L.1    Stahley, M.R.2    Kosek, A.B.3    Wang, J.4    Strobel, S.A.5
  • 2
    • 0242500997 scopus 로고    scopus 로고
    • Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA
    • Bartley, L.E., Zhuang, X., Das, R., Chu, S., and Herschlag, D. 2003. Exploration of the transition state for tertiary structure formation between an RNA helix and a large structured RNA. J. Mol. Biol. 328: 1011-1026.
    • (2003) J. Mol. Biol. , vol.328 , pp. 1011-1026
    • Bartley, L.E.1    Zhuang, X.2    Das, R.3    Chu, S.4    Herschlag, D.5
  • 3
    • 0026678180 scopus 로고
    • Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods
    • Bevilacqua, P.C., Kierzek, R., Johnson, K.A., and Turner, D.H. 1992. Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods. Science 258: 1355-1357.
    • (1992) Science , vol.258 , pp. 1355-1357
    • Bevilacqua, P.C.1    Kierzek, R.2    Johnson, K.A.3    Turner, D.H.4
  • 5
    • 0028027336 scopus 로고
    • Fluorescence-detected stopped-flow with a pyrene-labeled substrate reveals that guanosine facilitates docking of the 5′ cleavage site into a high free-energy binding mode in the Tetrahymena ribozyme
    • Bevilacqua, P.C., Li, Y., and Turner, D.H. 1994. Fluorescence-detected stopped-flow With a pyrene-labeled substrate reveals that guanosine facilitates docking of the 5′ cleavage site into a high free-energy binding mode in the Tetrahymena ribozyme. Biochemistry 33: 11340-11348.
    • (1994) Biochemistry , vol.33 , pp. 11340-11348
    • Bevilacqua, P.C.1    Li, Y.2    Turner, D.H.3
  • 6
    • 0030065737 scopus 로고    scopus 로고
    • A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme
    • Bevilacqua, P.C., Sugimoto, N., and Turner, D.H. 1996. A mechanistic framework for the second step of splicing catalyzed by the Tetrahymena ribozyme. Biochemistry 35: 648-658.
    • (1996) Biochemistry , vol.35 , pp. 648-658
    • Bevilacqua, P.C.1    Sugimoto, N.2    Turner, D.H.3
  • 7
    • 0024370698 scopus 로고
    • Selection of the 3′-splice site in group I introns
    • Burke, J.M. 1989. Selection of the 3′-splice site in group I introns. FEBS Lett. 250: 129-133.
    • (1989) FEBS Lett. , vol.250 , pp. 129-133
    • Burke, J.M.1
  • 8
    • 0030015953 scopus 로고    scopus 로고
    • An RNA conformational change between the two chemical steps of group II self-splicing
    • Chanfreau, G. and Jacquier, A. 1996. An RNA conformational change between the two chemical steps of group II self-splicing. EMBO J. 15: 3466-3476.
    • (1996) EMBO J. , vol.15 , pp. 3466-3476
    • Chanfreau, G.1    Jacquier, A.2
  • 9
    • 0027724213 scopus 로고
    • Self-splicing of the Tetrahymena pre-ribosomal-RNA is decreased by misfolding during transcription
    • Emerick, V.L. and Woodson, S.A. 1993. Self-splicing of the Tetrahymena pre-ribosomal-RNA is decreased by misfolding during transcription. Biochemistry 32: 14062-14067.
    • (1993) Biochemistry , vol.32 , pp. 14062-14067
    • Emerick, V.L.1    Woodson, S.A.2
  • 10
    • 0029800606 scopus 로고    scopus 로고
    • Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron
    • Emerick, V.L., Pan, J., and Woodson, S.A. 1996. Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron. Biochemistry 35: 13469-13477.
    • (1996) Biochemistry , vol.35 , pp. 13469-13477
    • Emerick, V.L.1    Pan, J.2    Woodson, S.A.3
  • 11
    • 0034646209 scopus 로고    scopus 로고
    • The P5abc peripheral element facilitates preorganization of the Tetrahymena group I ribozyme for catalysis
    • Engelhardt, M.A., Doherty, E.A., Knitt, D.S., Doudna, J.A., and Herschlag, D. 2000. The P5abc peripheral element facilitates preorganization of the Tetrahymena group I ribozyme for catalysis. Biochemistry 39: 2639-2651.
    • (2000) Biochemistry , vol.39 , pp. 2639-2651
    • Engelhardt, M.A.1    Doherty, E.A.2    Knitt, D.S.3    Doudna, J.A.4    Herschlag, D.5
  • 12
    • 0029880939 scopus 로고    scopus 로고
    • Conformational switches involved in orchestrating the successive steps of group I RNA splicing
    • Golden, B.L. and Cech, T.R. 1996. Conformational switches involved in orchestrating the successive steps of group I RNA splicing. Biochemistry 35: 3754-3763.
    • (1996) Biochemistry , vol.35 , pp. 3754-3763
    • Golden, B.L.1    Cech, T.R.2
  • 13
    • 0032500731 scopus 로고    scopus 로고
    • A preorganized active site in the crystal structure of the Tetrahymena ribozyme
    • Golden, B.L., Gooding, A.R., Podell, E.R., and Cech, T.R. 1998. A preorganized active site in the crystal structure of the Tetrahymena ribozyme. Science 282: 259-264.
    • (1998) Science , vol.282 , pp. 259-264
    • Golden, B.L.1    Gooding, A.R.2    Podell, E.R.3    Cech, T.R.4
  • 14
    • 0026580844 scopus 로고
    • Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme
    • Herschlag, D. 1992. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme. Biochemistry 31: 1386-1399.
    • (1992) Biochemistry , vol.31 , pp. 1386-1399
    • Herschlag, D.1
  • 15
    • 0025085590 scopus 로고
    • Catalysis of RNA cleavage by the Tetrahymena-thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active-site
    • Herschlag, D. and Cech, T.R. 1990. Catalysis of RNA cleavage by the Tetrahymena-thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active-site. Biochemistry 29: 10159-10171.
    • (1990) Biochemistry , vol.29 , pp. 10159-10171
    • Herschlag, D.1    Cech, T.R.2
  • 16
    • 0027181643 scopus 로고
    • Contributions of 2′-hydroxyl groups of the RNA substrate to binding and catalysis by the Tetrahymena ribozyme: An energetic picture of an activesite composed of RNA
    • Herschlag, D., Eckstein, F., and Cech, T.R. 1993. Contributions of 2′-hydroxyl groups of the RNA substrate to binding and catalysis by the Tetrahymena ribozyme: An energetic picture of an activesite composed of RNA. Biochemistry 32: 8299-8311.
    • (1993) Biochemistry , vol.32 , pp. 8299-8311
    • Herschlag, D.1    Eckstein, F.2    Cech, T.R.3
  • 17
    • 0037418272 scopus 로고    scopus 로고
    • Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: Implications for RNA structure and preorganization
    • Karbstein, K. and Herschlag, D. 2003. Extraordinarily slow binding of guanosine to the Tetrahymena group I ribozyme: Implications for RNA structure and preorganization. Proc. Natl. Acad. Sci. 100: 2300-2305.
    • (2003) Proc. Natl. Acad. Sci. , vol.100 , pp. 2300-2305
    • Karbstein, K.1    Herschlag, D.2
  • 18
    • 0037125926 scopus 로고    scopus 로고
    • Probing the Tetrahymena group I reaction in both directions
    • Karbstein, K., Carroll, K.S., and Herschlag, D. 2002. Probing the Tetrahymena group I reaction in both directions. Biochemistry 41: 11171-11183.
    • (2002) Biochemistry , vol.41 , pp. 11171-11183
    • Karbstein, K.1    Carroll, K.S.2    Herschlag, D.3
  • 20
    • 0028077048 scopus 로고
    • Dissection of the role of the conserved G-U pair in group I RNA self-splicing
    • Knitt, D.S., Narlikar, G.J., and Herschlag, D. 1994. Dissection of the role of the conserved G-U pair in group I RNA self-splicing. Biochemistry 33: 13864-13879.
    • (1994) Biochemistry , vol.33 , pp. 13864-13879
    • Knitt, D.S.1    Narlikar, G.J.2    Herschlag, D.3
  • 21
    • 0027170454 scopus 로고
    • Guanosine binding to the Tetrahymena ribozyme: Thermodynamic coupling with oligonucleotide binding
    • McConnell, T.S., Cech, T.R., and Herschlag, D. 1993. Guanosine binding to the Tetrahymena ribozyme: Thermodynamic coupling with oligonucleotide binding. Proc. Natl. Acad. Sci. 90: 8362-8366.
    • (1993) Proc. Natl. Acad. Sci. , vol.90 , pp. 8362-8366
    • McConnell, T.S.1    Cech, T.R.2    Herschlag, D.3
  • 22
    • 0029882895 scopus 로고    scopus 로고
    • Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: Insights into catalysis and the second step of self-splicing
    • Mei, R. and Herschlag, D. 1996. Mechanistic investigations of a ribozyme derived from the Tetrahymena group I intron: Insights into catalysis and the second step of self-splicing. Biochemistry 35: 5796-5809.
    • (1996) Biochemistry , vol.35 , pp. 5796-5809
    • Mei, R.1    Herschlag, D.2
  • 23
    • 0025678737 scopus 로고
    • Modeling of the three-dimensional architecture of group I catalytic introns based on comparative sequence-analysis
    • Michel, F. and Westhof, E. 1990. Modeling of the three-dimensional architecture of group I catalytic introns based on comparative sequence-analysis. J. Mol. Biol. 216: 585-610.
    • (1990) J. Mol. Biol. , vol.216 , pp. 585-610
    • Michel, F.1    Westhof, E.2
  • 24
    • 0024427177 scopus 로고
    • The guanosine binding-site of the Tetrahymena ribozyme
    • Michel, F., Hanna, M., Green, R., Bartel, D.P., and Szostak, J.W. 1989. The guanosine binding-site of the Tetrahymena ribozyme. Nature 342: 391-395.
    • (1989) Nature , vol.342 , pp. 391-395
    • Michel, F.1    Hanna, M.2    Green, R.3    Bartel, D.P.4    Szostak, J.W.5
  • 25
    • 0027281334 scopus 로고
    • Binding of guanosine and 3′ splice site analogs to a group I ribozyme: Interactions with functional groups of guanosine and with additional nucleotides
    • Moran, S., Kierzek, R., and Turner, D.H. 1993. Binding of guanosine and 3′ splice site analogs to a group I ribozyme: Interactions with functional groups of guanosine and with additional nucleotides. Biochemistry 32: 5247-5256.
    • (1993) Biochemistry , vol.32 , pp. 5247-5256
    • Moran, S.1    Kierzek, R.2    Turner, D.H.3
  • 26
    • 0029746472 scopus 로고    scopus 로고
    • Isolation of a local tertiary folding transition in the context of a globally folded RNA
    • Narlikar, G.J. and Herschlag, D. 1996. Isolation of a local tertiary folding transition in the context of a globally folded RNA. Nat. Struct. Biol. 3: 701-710.
    • (1996) Nat. Struct. Biol. , vol.3 , pp. 701-710
    • Narlikar, G.J.1    Herschlag, D.2
  • 27
    • 0032516432 scopus 로고    scopus 로고
    • Direct demonstration of the catalytic role of binding interactions in an enzymatic reaction
    • -. 1998. Direct demonstration of the catalytic role of binding interactions in an enzymatic reaction. Biochemistry 37: 9902-9911.
    • (1998) Biochemistry , vol.37 , pp. 9902-9911
  • 28
    • 0029008042 scopus 로고
    • Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization
    • Narlikar, G.J., Gopalakrishnan, V., McConnell, T.S., Usman, N., and Herschlag, D. 1995. Use of binding energy by an RNA enzyme for catalysis by positioning and substrate destabilization. Proc. Natl. Acad. Sci. 92: 3668-3672.
    • (1995) Proc. Natl. Acad. Sci. , vol.92 , pp. 3668-3672
    • Narlikar, G.J.1    Gopalakrishnan, V.2    McConnell, T.S.3    Usman, N.4    Herschlag, D.5
  • 29
    • 0031036847 scopus 로고    scopus 로고
    • Quantitating tertiary binding energies of 2′ OH groups on the P1 duplex of the Tetrahymena ribozyme: Intrinsic binding energy in an RNA enzyme
    • Narlikar, G.J., Khosla, M., Usman, N., and Herschlag, D. 1997. Quantitating tertiary binding energies of 2′ OH groups on the P1 duplex of the Tetrahymena ribozyme: Intrinsic binding energy in an RNA enzyme. Biochemistry 36: 2465-2477.
    • (1997) Biochemistry , vol.36 , pp. 2465-2477
    • Narlikar, G.J.1    Khosla, M.2    Usman, N.3    Herschlag, D.4
  • 30
    • 0033607247 scopus 로고    scopus 로고
    • Characterization of a local folding event of the Tetrahymena group I ribozyme: Effects of oligonucleotide substrate length, pH and temperature on the two substrate binding steps
    • Narlikar, G.J., Bartley, L.E., Khosla, M., and Herschlag, D. 1999. Characterization of a local folding event of the Tetrahymena group I ribozyme: Effects of oligonucleotide substrate length, pH and temperature on the two substrate binding steps. Biochemistry 38: 14192-14204.
    • (1999) Biochemistry , vol.38 , pp. 14192-14204
    • Narlikar, G.J.1    Bartley, L.E.2    Khosla, M.3    Herschlag, D.4
  • 31
    • 0037184536 scopus 로고    scopus 로고
    • Selection of tRNA by the ribosome requires a transition from an open to a closed form
    • Ogle, J.M., Murphy, F.V., Tarry, M.J., and Ramakrishnan, V. 2002. Selection of tRNA by the ribosome requires a transition from an open to a closed form. Cell 111: 721-732.
    • (2002) Cell , vol.111 , pp. 721-732
    • Ogle, J.M.1    Murphy, F.V.2    Tarry, M.J.3    Ramakrishnan, V.4
  • 32
    • 0033961143 scopus 로고    scopus 로고
    • Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome
    • Pape, T., Wintermeyer, W., and Rodnina, M.V. 2000. Conformational switch in the decoding region of 16S rRNA during aminoacyl-tRNA selection on the ribosome. Nat. Struct. Biol. 7: 104-107.
    • (2000) Nat. Struct. Biol. , vol.7 , pp. 104-107
    • Pape, T.1    Wintermeyer, W.2    Rodnina, M.V.3
  • 33
    • 0026427239 scopus 로고
    • Ribozyme recognition of RNA by tertiary interactions with specific ribose 2′-OH groups
    • Pyle, A.M. and Cech, T.R. 1991. Ribozyme recognition of RNA by tertiary interactions with specific ribose 2′-OH groups. Nature 350: 628-631.
    • (1991) Nature , vol.350 , pp. 628-631
    • Pyle, A.M.1    Cech, T.R.2
  • 34
    • 0032537739 scopus 로고    scopus 로고
    • RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2
    • Raghunathan, P.L. and Guthrie, C. 1998. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr. Biol. 8: 847-855.
    • (1998) Curr. Biol. , vol.8 , pp. 847-855
    • Raghunathan, P.L.1    Guthrie, C.2
  • 35
    • 0033520410 scopus 로고    scopus 로고
    • New pathways in folding of the Tetrahymena group I RNA enzyme
    • Russell, R. and Herschlag, D. 1999a. New pathways in folding of the Tetrahymena group I RNA enzyme. J. Mol. Biol. 291: 1155-1167.
    • (1999) J. Mol. Biol. , vol.291 , pp. 1155-1167
    • Russell, R.1    Herschlag, D.2
  • 36
    • 0033035042 scopus 로고    scopus 로고
    • Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme
    • -. 1999b. Specificity from steric restrictions in the guanosine binding pocket of a group I ribozyme. RNA 5: 158-166.
    • (1999) RNA , vol.5 , pp. 158-166
  • 37
    • 0033600571 scopus 로고    scopus 로고
    • Probing the role of metal ions in RNA catalysis: Kinetic and thermodynamic characterization of a metal ion interaction with the 2′-moiety of the guanosine nucleophile in the Tetrahymena group I ribozyme
    • Shan, S. and Herschlag, D. 1999. Probing the role of metal ions in RNA catalysis: Kinetic and thermodynamic characterization of a metal ion interaction with the 2′-moiety of the guanosine nucleophile in the Tetrahymena group I ribozyme. Biochemistry 38: 10958-10975.
    • (1999) Biochemistry , vol.38 , pp. 10958-10975
    • Shan, S.1    Herschlag, D.2
  • 38
    • 0036074219 scopus 로고    scopus 로고
    • Dissection of a metal-ion-mediated conformational change in Tetrahymena ribozyme catalysis
    • -. 2002. Dissection of a metal-ion-mediated conformational change in Tetrahymena ribozyme catalysis. RNA 8: 861-872.
    • (2002) RNA , vol.8 , pp. 861-872
  • 39
    • 0035340961 scopus 로고    scopus 로고
    • Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction
    • Shan, S., Kravchuk, A.V., Piccirilli, J.A, and Herschlag, D. 2001. Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction. Biochemistry 40: 5161-5171.
    • (2001) Biochemistry , vol.40 , pp. 5161-5171
    • Shan, S.1    Kravchuk, A.V.2    Piccirilli, J.A.3    Herschlag, D.4
  • 40
    • 0033010430 scopus 로고    scopus 로고
    • An RNA switch at the 5′ splice site requires ATP and the DEAD box protein Prp28p
    • Staley, J.P. and Guthrie, C. 1999. An RNA switch at the 5′ splice site requires ATP and the DEAD box protein Prp28p. Mol. Cell 3: 55-64.
    • (1999) Mol. Cell , vol.3 , pp. 55-64
    • Staley, J.P.1    Guthrie, C.2
  • 41
    • 0027767532 scopus 로고
    • Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme
    • Strobel, S.A. and Cech, T.R. 1993. Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme. Biochemistry 32: 13593-13604.
    • (1993) Biochemistry , vol.32 , pp. 13593-13604
    • Strobel, S.A.1    Cech, T.R.2
  • 42
    • 0028955347 scopus 로고
    • Minor-groove recognition of the conserved G-U pair at the Tetrahymena ribozyme reaction site
    • -. 1995. Minor-groove recognition of the conserved G-U pair at the Tetrahymena ribozyme reaction site. Science 267: 675-679.
    • (1995) Science , vol.267 , pp. 675-679
  • 43
    • 0030068393 scopus 로고    scopus 로고
    • Exocyclic amine of the conserved G-U pair at the cleavage site of the Tetrahymena ribozyme contributes to 5′-splice site selection and transition-state stabilization
    • -. 1996. Exocyclic amine of the conserved G-U pair at the cleavage site of the Tetrahymena ribozyme contributes to 5′-splice site selection and transition-state stabilization. Biochemistry 35: 1201-1211.
    • (1996) Biochemistry , vol.35 , pp. 1201-1211
  • 45
    • 0037176844 scopus 로고    scopus 로고
    • Identification of an active site ligand for a group I ribozyme catalytic metal ion
    • Szewczak, A.A., Kosek, A.B., Piccirilli, J.A., and Strobel, S.A. 2002. Identification of an active site ligand for a group I ribozyme catalytic metal ion. Biochemistry 41: 2516-2525.
    • (2002) Biochemistry , vol.41 , pp. 2516-2525
    • Szewczak, A.A.1    Kosek, A.B.2    Piccirilli, J.A.3    Strobel, S.A.4
  • 46
    • 0031025796 scopus 로고    scopus 로고
    • Joining the two domains of a group I ribozyme to form the catalytic core
    • Tanner, M.A. and Cech, T.R. 1997. Joining the two domains of a group I ribozyme to form the catalytic core. Science 275: 847-849.
    • (1997) Science , vol.275 , pp. 847-849
    • Tanner, M.A.1    Cech, T.R.2
  • 47
    • 0030866522 scopus 로고    scopus 로고
    • Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes
    • Tanner, M.A., Anderson, E.M., Guteil, R.R., and Cech, T.R. 1997. Mutagenesis and comparative sequence analysis of a base triple joining the two domains of group I ribozymes. RNA 3: 1037-1051.
    • (1997) RNA , vol.3 , pp. 1037-1051
    • Tanner, M.A.1    Anderson, E.M.2    Guteil, R.R.3    Cech, T.R.4
  • 48
    • 0025205075 scopus 로고
    • The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification
    • Walstrum, S.A. and Uhlenbeck, O.C. 1990. The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification. Biochemistry 29: 10573-10576.
    • (1990) Biochemistry , vol.29 , pp. 10573-10576
    • Walstrum, S.A.1    Uhlenbeck, O.C.2
  • 49
    • 0025975586 scopus 로고
    • Alternative secondary structures in the 5′ exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA
    • Woodson, S.A. and Cech, T.R. 1991. Alternative secondary structures in the 5′ exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA. Biochemistry 30: 2042-2050.
    • (1991) Biochemistry , vol.30 , pp. 2042-2050
    • Woodson, S.A.1    Cech, T.R.2
  • 50
    • 0024285808 scopus 로고
    • Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: Enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme substrate complexes
    • Zaug, A.J., Grosshans, C.A., and Cech, T.R. 1988. Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: Enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme substrate complexes. Biochemistry 27: 8924-8931.
    • (1988) Biochemistry , vol.27 , pp. 8924-8931
    • Zaug, A.J.1    Grosshans, C.A.2    Cech, T.R.3


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.