title: Detailed analysis of base preferences at the cleavage site of a trans-acting HDV ribozyme: a mutation that changes cleavage site specificity.
abstract: In our previous attempt at in vitro selection of a trans - acting human hepatitis delta virus (HDV) ribozyme, we found that one of the variants, G10-68-725G, cleaved a 13 nt substrate, HDVS1, at two sites [Nishikawa,F., Kawakami,J., Chiba,A., Shirai,M., Kumar,P.K.R. and Nishikawa,S. (1996) Eur. J. Biochem., 237, 712-718]. One site was the normal cleavage site and the other site was shifted 1 nt toward the 3'-end. To clarify the interactions between nucleotides around the cleavage site of the trans -acting HDV ribozyme, we analyzed the efficiency of the reaction for every possible base pair between the substrate and the ribozyme at positions -1 (-1N:726N) and +1 (+1N:725N) relative to the cleavage site using the genomic HDV ribozyme, TdS4(Xho), and derivatives of the most active variant, G10-68. These mutagenesis analyses revealed that the +1 base of the substrate affects the structure of the catalytic core in the complex with G10-68-725G, substrate and divalent metal ions, and it shifts the cleavage site. In a comparison with other variants of the trans -acting HDV ribozyme, we found that this cleavage site shift occurred only with G10-68-725G.
authors.affiliation: 1 National Institute of Bioscience and Human Technology, AIST, MITI, 1-1 Higashi, Tsukuba Science City, Ibaraki 305, Japan. Japan.
authors.forename: F
authors.initials: F
authors.lastname: Nishikawa
authors.forename: H
authors.initials: H
authors.lastname: Fauzi
authors.forename: S
authors.initials: S
authors.lastname: Nishikawa
mesh_terms: D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D014644:Genetic Variation; D003698:Hepatitis Delta Virus / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D008961:Models, Structural; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity; D014158:Transcription, Genetic
chemical_list: D009843:Oligoribonucleotides; D016337:RNA, Catalytic