Cross-linking experiments reveal the presence of novel structural features between a hepatitis delta virus ribozyme and its substrate.
The kinetic pathway of a trans-acting delta ribozyme includes an essential structural rearrangement involving the P1 stem, a stem that is formed between the substrate and the ribozyme. We performed cross-linking experiments to determine the substrate position within the catalytic center of an antigenomic, trans-acting, delta ribozyme. Substrates that included a 4-thiouridine either in position -1, +4, or +8 (i.e., adjacent to the cleavage site, or located either in the middle of or at the 3'-end of the P1 stem, respectively) were synthesized and shown to be efficiently cleaved. Examination of the cross-linking conditions, the use of various mutated ribozymes, as well as the probing and characterization of the resulting ribozyme-substrate complexes, revealed several new features of the molecular mechanism: (1) the close proximity of several bases between nucleotides of the substrate and ribozyme; (2) the active ribozyme-substrate complex folds in a manner that docks the middle of the P1 stem on the P3 stem, while concomitantly the scissile phosphate is in close proximity to the catalytic cytosine; and, (3) some complexes appear to be compatible with being active intermediates along the folding pathway, while others seem to correspond to misfolded structures. To provide a model representation of these data, a three-dimensional structure of the delta ribozyme was developed using several RNA bioinformatic software packages.
Ouellet
Jonathan
J
RNA group/groupe ARN, Département de biochimie, Faculté de médecine, Université de Sherbrooke, Québec J1H 5N4, Canada.
Perreault
Jean-Pierre
JP
D001483:Base Sequence; D003432:Cross-Linking Reagents; D003698:Hepatitis Delta Virus / Q000235:genetics*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics
D003432:Cross-Linking Reagents; D016337:RNA, Catalytic; D012367:RNA, Viral