title: Investigation of Mg2+- and temperature-dependent folding of the hairpin ribozyme by photo-crosslinking: effects of photo-crosslinker tether length and chemistry.
abstract: We have used photo-crosslinking to investigate the structure and dynamics of four-way junction hairpin ribozyme constructs. Four phenylazide photo-crosslinkers were coupled to 2'-NH2-modified U+2 in the substrate and irradiated at different Mg2+ concentrations and temperatures. Consistent with the role of divalent metal ions in hairpin ribozyme folding, we observed more interdomain crosslinks in the presence of Mg2+ than in its absence. In general, we observed intradomain crosslinks to nucleotides 2-11 and interdomain crosslinks to the U1A binding loop. Crosslinks to A26 and G36 in domain B were also observed when crosslinking was carried out at -78 degrees C. In contrast to crosslinking results at higher temperatures (0, 25 and 37 degrees C), similar crosslinks were obtained in the presence and absence of Mg2+ at -78 degrees C, suggesting Mg2+ stabilizes a low-energy hairpin ribozyme conformation. We also evaluated the effects of photo-crosslinker structure and mechanism on crosslinks. First, most crosslinks were to unpaired nucleotides. Second, shorter and longer photo-crosslinkers formed crosslinks to intradomain locations nearer to and farther from photo-crosslinker modification, respectively. Finally, fluorine substitutions on the phenylazide ring did not change the locations of crosslinks, but rather decreased crosslinking efficiency. These findings have implications for the use of phenylazide photo-crosslinkers in structural studies of RNA.
authors.affiliation: Department of Chemistry, University of Washington Seattle, WA 98195-1700, USA.
authors.forename: Emily J
authors.initials: EJ
authors.lastname: Borda
authors.forename: Snorri Th
authors.initials: ST
authors.lastname: Sigurdsson
mesh_terms: D001386:Azides / Q000737:chemistry*; D001483:Base Sequence; D002413:Cations, Divalent; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry*; D008027:Light; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D013696:Temperature
chemical_list: D001386:Azides; D002413:Cations, Divalent; D003432:Cross-Linking Reagents; D016337:RNA, Catalytic; C418481:hairpin ribozyme; C014747:phenylazide; D008274:Magnesium
references.citation: Protein Expr Purif. 1997 Feb;9(1):142-51
references.pmid: 9116496
references.citation: Nature. 2001 Apr 12;410(6830):780-6
references.pmid: 11298439
references.citation: Nat Struct Biol. 1999 Mar;6(3):212-6
references.pmid: 10074938
references.citation: J Am Chem Soc. 2001 Mar 7;123(9):1951-62
references.pmid: 11456816
references.citation: Biochemistry. 1989 Jun 13;28(12):4929-33
references.pmid: 2765519
references.citation: J Mol Biol. 1999 Mar 26;287(2):239-51
references.pmid: 10080888
references.citation: Chem Biol. 1997 Jul;4(7):513-7
references.pmid: 9263639
references.citation: J Org Chem. 1996 May 31;61(11):3883-3884
references.pmid: 11667245
references.citation: J Mol Biol. 2004 Feb 6;336(1):69-79
references.pmid: 14741204
references.citation: Biochemistry. 2001 Nov 20;40(46):13849-56
references.pmid: 11705374
references.citation: RNA. 2002 May;8(5):587-600
references.pmid: 12022226
references.citation: Biopolymers. 2004 Jan;73(1):71-8
references.pmid: 14691941
references.citation: J Mol Biol. 2000 Mar 24;297(2):269-91
references.pmid: 10715200
references.citation: Chem Biol. 1997 Aug;4(8):619-30
references.pmid: 9281529
references.citation: RNA. 2000 Dec;6(12):1727-36
references.pmid: 11142373
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9308-13
references.pmid: 12883002
references.citation: Nucleic Acids Res. 1996 Mar 1;24(5):977-8
references.pmid: 8600468
references.citation: Nucleic Acids Res. 2002 Mar 15;30(6):1287-91
references.pmid: 11884625
references.citation: Nucleic Acids Res. 1992 Sep 11;20(17):4507-13
references.pmid: 1383927
references.citation: Biochemistry. 1997 Apr 1;36(13):3930-40
references.pmid: 9092823
references.citation: J Mol Biol. 1997 Nov 28;274(2):197-212
references.pmid: 9398527
references.citation: Nucleic Acids Res. 1997 Oct 1;25(19):3760-6
references.pmid: 9380495
references.citation: Biochemistry. 1999 Aug 24;38(34):11040-50
references.pmid: 10460159
references.citation: Biochemistry. 1998 Dec 15;37(50):17629-36
references.pmid: 9860879
references.citation: Biophys J. 2004 Jul;87(1):457-67
references.pmid: 15240479
references.citation: J Am Chem Soc. 2003 Sep 10;125(36):10850-61
references.pmid: 12952464
references.citation: Bioconjug Chem. 1993 Mar-Apr;4(2):178-83
references.pmid: 7873650
references.citation: Nucleic Acids Res. 1996 Apr 1;24(7):1351-9
references.pmid: 8614641
references.citation: Bioconjug Chem. 1993 Mar-Apr;4(2):172-7
references.pmid: 7873649
references.citation: Nucleic Acids Res. 1996 Aug 15;24(16):3129-33
references.pmid: 8774891
references.citation: Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9302-7
references.pmid: 12869691
references.citation: Biochemistry. 1996 May 14;35(19):6026-36
references.pmid: 8634244
references.citation: Biochimie. 1999 Mar;81(3):201-12
references.pmid: 10385001
references.citation: EMBO J. 2002 Sep 2;21(17):4691-8
references.pmid: 12198171