Effects of circular permutation on the cis-cleavage reaction of a hepatitis delta virus ribozyme: application to trans-acting ribozyme design.
In this study we investigated the effects of the relocation of the wild type termini on the folding of a cis-cleaving RNA Rz 1 that was modified from the autolytic domains of hepatitis delta virus (HDV) RNA. Ten circularly permuted (CP) isomers of this ribozyme were synthesized. The structure homogeneity of RNA molecules, the molar ratio of the active species that undergoes cis-cleavage, and the rate of cis-cleavage were examined for each construct. CP isomers with new termini in H1 or at the junction of H2-H3, H1-J1/4, J1/4-H4, or H4-J4/2 of the proposed pseudoknot-like structure were inactive. The single breaks of phosphodiester bond in H2, J1/2, Lp4, and at the 3'-end of Lp3 decreased but did not abolish autolytic activity. The structural heterogeneity of RNA molecules may account for the limited cis-cleavage of the latter three isomers. The findings of circular permutation analysis were used as the basis for designing an active trans-cleaving ribozyme by dividing the cis-cleaving ribozyme into two subdomains at J1/2 and Lp4. The ribozyme subdomain catalyzed the site-specific cleavage of the circularly permuted composite substrate RNA in trans. Thus, the structure of HDV autolytic domain could be re-formed after two subdomains were associated through the base-pairing interactions of H1, H2, and H4.
Lai
Y C
YC
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan.
Lee
J Y
JY
Liu
H J
HJ
Lin
J Y
JY
Wu
H N
HN
D001483:Base Sequence; D017931:DNA Primers; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D013698:Templates, Genetic; D013997:Time Factors
D017931:DNA Primers; D016337:RNA, Catalytic; D012367:RNA, Viral