The hairpin ribozyme substrate binding-domain: a highly constrained D-shaped conformation.
The two domains of the hairpin ribozyme-substrate complex, usually depicted as straight structural elements, must interact with one another in order to form an active conformation. Little is known about the internal geometry of the individual domains in an active docked complex. Using various crosslinking and structural approaches in conjunction with molecular modeling (constraint-satisfaction program MC-SYM), we have investigated the conformation of the substrate-binding domain in the context of the active docked ribozyme-substrate complex. The model generated by MC-SYM showed that the domain is not straight but adopts a bent conformation (D-shaped) in the docked state of the ribozyme, indicating that the two helices bounding the internal loop are closer than was previously assumed. This arrangement rationalizes the observed ability of hairpin ribozymes with a circularized substrate-binding strand to cleave a circular substrate, and provides essential information concerning the organization of the substrate in the active conformation. The internal geometry of the substrate-binding strand places G8 of the substrate-binding strand near the cleavage site, which has allowed us to predict the crucial role played by this nucleotide in the reaction chemistry.
Pinard
R
R
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington, VT 05405, USA.
Lambert
D
D
Heckman
J E
JE
Esteban
J A
JA
Gundlach
C W
CW
Hampel
K J
KJ
Glick
G D
GD
Walter
N G
NG
Major
F
F
Burke
J M
JM
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D016337:RNA, Catalytic; D012367:RNA, Viral; C418481:hairpin ribozyme