A NEW-TYPE OF HAIRPIN RIBOZYME CONSISTING OF 3 DOMAINS

We have constructed a new hairpin ribozyme with three stem-loop domain s. 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 autop rocessing activity was proved to be derived from the catalytic reactio n, similar to the wild-type ribozyme. Furthermore, the structure of th e 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 de rived from the mRNA of the human inducible nitric oxide synthase and w as 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 ribozym e thus obtained has three stem-loop domains and is able to act as a ca talytic RNA for both cis and trans cleavage. These ribozymes are of in terest from the point of the structure-function relationship of the ha irpin ribozyme and provide an important insight into over understandin g of the role of the domain-domain interaction in the catalytic activi ty.