Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes.
The complete VS ribozyme comprises seven helical segments, connected by three three-way RNA junctions. In the presence of Mg(2+) ions, cleavage occurs within the internal loop of helix I. This requires the participation of a guanine (G638) within the helix I loop, and a remote adenine (A756) within an internal loop of helix VI. Previous structural studies have suggested that helix I docks into the fold of the remaining part of the ribozyme, bringing A756 and G638 close to the scissile phosphate to allow the cleavage reaction to proceed. We show here that while either A756C or G638A individually exhibit very low cleavage activity, a mixture of the two variants leads to cleavage of the A756C RNA, but not the G638A RNA. The rate of cleavage depends on the concentration of the VS G638A RNA, as expected for a bimolecular interaction. This regaining of cleavage activity by complementation indicates that helix I of one VS RNA can interact with another VS RNA molecule to generate a functional active site in trans.
Ouellet
Jonathan
J
Cancer Research UK Nucleic Acid Structure Research Group, The University of Dundee, Dundee, United Kingdom.
Byrne
Max
M
Lilley
David M J
DM
D001483:Base Sequence; D020134:Catalytic Domain; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic
Biochemistry. 2006 Nov 7;45(44):13394-400
17073461
Biochemistry. 2004 Feb 3;43(4):1118-25
14744158
EMBO J. 2002 May 15;21(10):2461-71
12006498
Structure. 2008 Sep 10;16(9):1357-67
18786398
EMBO J. 2007 May 16;26(10):2489-500
17464286
EMBO J. 1995 Jan 16;14(2):368-76
7835347
Nat Struct Biol. 1999 Jun;6(6):544-9
10360357
J Mol Biol. 2002 Jul 12;320(3):443-54
12096902
J Mol Biol. 2001 Sep 28;312(4):663-74
11575922
Mol Cell. 1998 May;1(6):873-81
9660970
Nat Struct Biol. 2003 Sep;10(9):708-12
12881719
EMBO J. 1996 Jun 3;15(11):2820-5
8654379
Cell. 1990 May 18;61(4):685-96
2160856
J Mol Biol. 2002 Oct 11;323(1):23-34
12368096
EMBO J. 2001 Mar 15;20(6):1415-24
11250907
RNA. 2008 Apr;14(4):736-48
18314503
RNA. 2004 May;10(5):880-8
15100442
Nucleic Acids Res. 2001 Dec 15;29(24):5115-20
11812844
Proc Natl Acad Sci U S A. 2007 Apr 3;104(14):5818-23
17389378
Proc Natl Acad Sci U S A. 1995 May 9;92(10):4686-90
7753865