Identification of important bases in a single‐stranded region (SSrC) of the hepatitis delta (δ) virus ribozyme

• Published in: European journal of biochemistry Vol. 217; no. 1; pp. 29 - 36
• Main Authors: KAWAKAMI, Junji, KUMAR, P. K. R., SUH, Young‐Ah, NISHIKAWA, Fumiko, KAWAKAMI, Keiko, TAIRA, Kazunari, OHTSUKA, Eiko, NISHIKAWA, Satoshi
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.1993; Blackwell
• Subjects: Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; DNA - chemistry; Fundamental and applied biological sciences. Psychology; Hepatitis Delta Virus - genetics; Molecular Sequence Data; Nucleic Acid Conformation; Nucleic acids; Point Mutation; Polymerase Chain Reaction; RNA Splicing; RNA, Catalytic - chemistry; Rna, ribonucleoproteins; RNA, Viral - chemistry; Virus; Delta agent; Ribozyme; Structure activity relation; Site directed mutagenesis; Catalyst activity; In vitro
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1993.tb18214.x

Models for the secondary structure of genomic and antigenomic self‐cleaving RNAs of human hepatitis delta (δ) virus (HDV) have been proposed by several groups. Our recent results support a pseudoknot structure and have allowed us to identify functionally important nucleotides in single‐stranded regions [nucleotides 726–731 (SSrA) and nucleotides 762–766 (SSrB)]. For the identification of the important residues in the remaining single‐stranded region, nucleotides 708–715 (SSrC), of the genomic HDV ribozyme, we made derivatives with a single‐base substitution in the SSrC region. To screen inactive mutants rapidly, we use a simplified in‐vitro selection method. Among the various base substitutions in mutants in the SSrC, U708A, C709(A/G/U) and G713C variants had less than 10% of the cleavage activity of the wild‐type SSrC (HDV86). By analyzing the self‐cleavage activities of various mutants, we determined the base requirements for SSrC as 5′‐(U/C/G)‐C‐N‐N‐(C/A/G)‐(G/A/U)‐N‐N‐3′.