Structural basis for the guanosine requirement of the hairpin ribozyme.
To form a catalytically active complex, the essential nucleotides of the hairpin ribozyme, embedded within the internal loops of the two domains, must interact with one another. Little is known about the nature of these essential interdomain interactions. In the work presented here, we have used recent topographical constraints and other biochemical data in conjunction with molecular modeling (constraint-satisfaction program MC-SYM) to generate testable models of interdomain interactions. Visual analysis of the generated models has revealed a potential interdomain base pair between the conserved guanosine immediately downstream of the reactive phosphodiester (G(+1)) and C(25) within the large domain. We have tested this former model through activity assays, using all 16 combinations of bases at positions +1 and 25. When the standard ribozyme was used, catalytic activity was severely suppressed with substrates containing U(+1), C(+1), or A(+1). Similarly, mutations of the putative pairing partner (C(25) to A(25) or G(25)) reduce activity by several orders of magnitude. The U(25) substitution retains a significant level of activity, consistent with the possible formation of a G.U wobble pair. Strikingly, when combinations of Watson-Crick (or wobble) base pairs were introduced in these positions, catalytic activity was restored, strongly suggesting the existence of the proposed interaction. These results provide a structural basis for the guanosine requirement of this ribozyme and indicate that the hairpin ribozyme can now be engineered to cleave a wider range of RNA sequences.
Pinard
R
R
Department of Microbiology and Molecular Genetics, University of Vermont, Burlington 05405, USA.
Lambert
D
D
Walter
N G
NG
Heckman
J E
JE
Major
F
F
Burke
J M
JM
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003596:Cytosine / Q000737:chemistry; D006151:Guanosine / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000201:enzymology / Q000235:genetics; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D018749:RNA, Plant / Q000737:chemistry; D012984:Software; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry
D016337:RNA, Catalytic; D018749:RNA, Plant; D013873:Thionucleotides; D006151:Guanosine; D003596:Cytosine