ABSTRACT
Functional structures of hairpin ribozymes have been investigated by constructing various chemically modified molecules. Domain-exchange and linker insertion experiments were performed to find active conformations of the RNA enzyme showing cleavage activity. Our experiments and other evidence suggest that the active structure has a bent conformation, and that domain-interactions are essential for the cleavage activity.
Subject(s)
Nucleic Acid Conformation , RNA, Catalytic/chemistry , RNA, Viral/chemistry , Nepovirus/genetics , RNA/chemistry , RNA/metabolism , RNA, Catalytic/metabolism , RNA, Viral/metabolism , Substrate Specificity , Transcription, GeneticABSTRACT
We have constructed a new hairpin ribozyme with three stem-loop domains. In the ribozyme, another domain (domain I') was connected to the 3'-end of domain II of the parent hairpin ribozyme, and the new ribozyme can be trimmed after transcription from the DNA template using T7 RNA polymerase. Since a mutant ribozyme containing a substitution at the essential base in domain I' lacked the 3'-trimming reaction, the autoprocessing activity was proved to be derived from the catalytic reaction, similar to the wild-type ribozyme. Furthermore, the structure of the cleavage site from the self-trimming reaction was identified as a 2',3'-cyclic phosphate, which is the same as that of the wild-type. The processed ribozyme was designed to cleave an external substrate RNA derived from the mRNA of the human inducible nitric oxide synthase and was proved to cleave at the expected, unique site. The hairpin ribozyme containing the three-domains exhibited the 3'-self-trimming activity even in a runoff transcription reaction from the plasmid harboring the ribozyme gene with the three domains. The new type of hairpin ribozyme thus obtained has three stem-loop domains and is able to act as a catalytic RNA for both cis and trans cleavage. These ribozymes are of interest from the point of the structure-function relationship of the hairpin ribozyme and provide an important insight into over understanding of the role of the domain-domain interaction in the catalytic activity.
Subject(s)
Nucleic Acid Conformation , RNA, Catalytic/chemical synthesis , RNA, Catalytic/metabolism , RNA/chemistry , Base Sequence , RNA/genetics , RNA/metabolism , RNA, Catalytic/genetics , Substrate Specificity , Transcription, GeneticABSTRACT
Reversely joined ribozymes (Komatsu et al., 1995) have been proven to be active. Here we describe the construction of hairpin ribozymes with separated domains, but containing complementary arms for association of the two domains. Linker nucleotides were inserted between the arms and domains. These ribozymes were active under the standard conditions (12 mM MgCl2), depending on the length of the linker. When the complementary arms were covalently joined through a stable loop, these ribozymes showed cleavage activities. However, the K(m) value of the stem-loop ribozymes was found to be larger than that of the parent ribozyme, which can adopt both linear and bent conformations. Kinetic analyses of these modified hairpin ribozymes suggest a higher turnover of the hairpin ribozyme as compared to other small ribozymes. The present ribozymes provide insight into the nature of the domain interaction and are suitable for physicochemical studies on the tertiary structure of the hairpin ribozyme.
Subject(s)
Nucleic Acid Conformation , RNA, Catalytic/chemistry , RNA, Catalytic/metabolism , Base Sequence , Electrophoresis, Polyacrylamide Gel , Molecular Sequence Data , RNA/chemical synthesis , RNA/metabolism , Structure-Activity RelationshipABSTRACT
Hairpin ribozymes consist of two stem-loop domains, and these domains are assumed to interact with each other to produce the self-cleavage activity. We have studied the relationship of the tertiary structure of the hairpin ribozyme and the cleavage activity by dividing and re-joining the domains. A hairpin ribozyme (E50) was divided at the hinge region, and the main part was joined to a substrate (S1) using tri- or penta-cytidylates. These ribozymes retained the cleavage activity in the presence of the rest of the molecule, indicating that the active conformation could be maintained if the two domains interacted with each other. Based on the these results, we designed a new type of hairpin ribozyme by replacing one of the domains. To maintain the interaction of the domains, oligocytidylates were inserted at a junction. These reversely jointed ribozyme complexes showed cleavage activity that was dependent on the linker lengths. These modifications in the primary structure of the hairpin ribozyme confirm the structural requirement for the catalytic reaction and provide information for the correlation of the tertiary structure with the cleavage of the hairpin ribozyme.
Subject(s)
Nucleic Acid Conformation , RNA, Catalytic/chemistry , Base Sequence , Hydrogen Bonding , Kinetics , Molecular Sequence Data , RNA/chemical synthesis , RNA/chemistry , RNA, Catalytic/metabolism , Structure-Activity RelationshipABSTRACT
We have constructed a new type of hairpin ribozyme by cleaving and reverse-joining one of the two catalytic domains to conserve an essential bending structure. The two domains of the new ribozymes were tethered by different lengths of cytidylate linkers. These ribozymes retained the cleavage activity and the cleavage activities depended on the linker lengths. Although these rejoined ribozymes showed weak turnover abilities, those with 18 cytidylates showed a larger kcat/K(m) value than the natural hairpin ribozymes. These modifications in the primary structure of the hairpin ribozyme confirm the bent conformation for the catalytic reaction of this ribozyme, and provide a new approach for the design of highly efficient ribozymes.
