The solvent-protected core of the hairpin ribozyme-substrate complex.
The complex between the hairpin ribozyme and its substrate consists of two domains that must interact in order to form a catalytic complex, yet experimental evidence concerning the points of interaction between the two domains has been lacking. Here, we report the use of hydroxyl radical footprinting to define the interface between the two domains. Cations that support very efficient ribozyme catalysis (magnesium and cobalt(III) hexammine) lead to the formation of a docked complex that features several regions of protection, indicating a solvent-inaccessible core within the tertiary structure of the complex. Cations that are suboptimal in cleavage reactions do not produce complexes with regions of reduced solvent accessibility. Nucleotides encompassing the substrate cleavage site (c-2, a-1, g+1, and u+2) are strongly protected, suggesting their internalization into the catalytic core. Four distinct segments of the ribozyme are protected, including G11-A14, C25-C27, A38, and U42-A43. Protection of these sites is eliminated when g+1, an essential base at the cleavage site, is replaced by A. In addition, mutations which are known to decrease the fraction of docked complexes decrease or eliminate formation of a solvent-inaccessible core. Taken together, these observations demonstrate that we have identified the catalytic core of the active hairpin ribozyme-substrate complex.
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405, USA.
K J
KJ
Hampel
N G
NG
Walter
J M
JM
Burke
D001483:Base Sequence; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004952:Esters; D006868:Hydrolysis; D017665:Hydroxyl Radical; D046911:Macromolecular Substances; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*; D012997:Solvents; D013379:Substrate Specificity
D004952:Esters; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D016337:RNA, Catalytic; D012997:Solvents; C030862:cobalt ammonium complex; D017665:Hydroxyl Radical; D003035:Cobalt; D008274:Magnesium