Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA

Wu, Hurng‐Sheng; Huang, Zhi-Shun

doi:10.1093/nar/20.22.5937

Cited by 39 publications

(55 citation statements)

References 15 publications

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“…U pair in the HDV ribozymes is at the base of PI and is probably significant because of its proximity to the cleavage site. Replacing either or both of the bases that form the G.U pair results in decreased activity in both the genomic (Wu et al, 1993) and antigenomic sequences (Perrotta and Been, 1996;Wu and Huang, 1992). G.U and A .…”

Section: P1mentioning

confidence: 99%

Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Been

Wickham

1997

European Journal of Biochemistry

147

170

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Hepatitis delta virus (HDV) is a small single-stranded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self-cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The selfcleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling-circle replication to unit-length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysishas not yet been proven. A minimal natural ribozyme sequence with proficient in viiro self-cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (Pl-P4).The two pairings that define the 5' and 3' boundaries of the ribozyme, PI and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo-cross-linking, mutagenesis and computer-assisted modeling provides clues to the three-dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5' end of PI, is in close proximity to three singlestranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans-acting ribozymes and smaller more active cis-acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.

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Section: P1mentioning

confidence: 99%

Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Been

Wickham

1997

European Journal of Biochemistry

147

170

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“…1). Although, several investigations have been performed to address the questions related to the substrate specificity of ␦ ribozymes in both the cis-and transacting forms (2,(5)(6)(7)(8)(9)(10)(11)(12), most, if not all, experiments were carried out by randomly changing the base pairing combinations or by introducing mismatches which interfere with the WatsonCrick base pairing between the substrate and the ribozyme in the P1 stem (Fig. 1).…”

mentioning

confidence: 99%

“…One or two nucleotide mismatches at any position of the P1 stem, except positions 5 and 11 (numbering according to Fig. 1), completely destroyed the activity (2,(5)(6)(7)(8)(9)(10)(11)(12). Although these are composite results from various versions of ␦ ribozymes, these findings could be interpreted as indicating that the positions located at both extremities of the base paired stem formed by the substrate and the ribozyme were more likely to tolerate a mismatch, resulting in distortion of the P1 stem, than the internal positions.…”

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confidence: 99%

Substrate Specificity of δ Ribozyme Cleavage

Ananvoranich¹,

Perreault

1998

Journal of Biological Chemistry

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The specificity of ␦ ribozyme cleavage was investigated using a trans-acting antigenomic ␦ ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min ؊1 , an apparent K m of 17.9 nM and an apparent second-order rate constant of 1.89 ؋ 10 7 min ؊1 M ؊1 . The substrate specificity of the ␦ ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of ␦ ribozyme cleavage.␦ ribozymes derived from the genome of hepatitis ␦ virus (HDV) 1 are metalloenzymes. Like other catalytically active ribozymes, namely hammerhead and hairpin ribozymes, the ␦ ribozymes cleave a phosphodiester bond of their RNA substrates and give rise to reaction products containing a 5Ј-hydroxyl and a 2Ј,3Ј-cyclic phosphate termini. Two forms of ␦ ribozymes, namely genomic and antigenomic, were derived and referred to by the polarity of the HDV genome from which the ribozyme was generated. Both ␦ ribozyme forms exhibit selfcleavage activity, and it has been suggested that they are involved in the process of viral replication (1). This type of activity has been described as cis-acting ␦ ribozymes (2).Like other ribozymes, ␦ ribozymes have a potential application in gene therapy in which an engineered ribozyme is directed to inhibit gene expression by targeting a specific mRNA molecule. It has been demonstrated that a very low concentration (Ͻ0.1 mM) of Ca 2ϩ and Mg 2ϩ is required for ␦ ribozyme cleavage (3). ␦ ribozymes have a unique characteristic in their substrate binding, namely that only the 3Ј-portion of the substrate is required for binding to the ribozyme. A short stretch of nucleotides (7 nt) located on the substrate is required for cleavage. Although one might suspect the specificity of ␦ ribozyme cleavages due to their short recognition site, we view this characteristic of the ␦ ribozyme as an advantage for the future development of a therapeutic means of controlling, for example, a viral infection.Since little is known about the kinetic properties of ␦ ribozymes, study of the trans-acting system will enable us to answer some basic questions on both the structure required and the kinetic properties, including the substrate specificity, of ␦ ribozymes. Depending on the predicted secondary st...

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“…Both comparative sequence analysis and a variety of biochemical studies indicate that the genomic and antigenomic ribozymes have closely related, but not identical, structures. Several models for each ribozyme have been proposed, tested, refined (14,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) and reviewed (13). Despite the progress that has been made, more work is needed to establish the details ofthe active structures.…”

Section: Introductionmentioning

confidence: 99%

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones

Branch

Polaskova

1995

Nucl Acids Res

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We mapped 359 mutations at 25 positions in synthetic variants of the antigenomic ribozyme of the hepatitis delta agent by analyzing the sequences of 188 cDNA clones. These data were used to identify three features of the ribozyme: highly conserved nucleotides, positions with restricted nucleotide substitutions and three-dimensional relationships between nucleotides. The distribution of mutations at the 25 positions was as follows: G-11 (the eleventh nucleotide from the cleavage site) was mutated in 56 clones; G-12 in 36;

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Mutagenesis analysis of the self-cleavage domain of hepatitis delta virus antigenomic RNA

Cited by 39 publications

References 15 publications

Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Substrate Specificity of δ Ribozyme Cleavage

3-D models of the antigenomic ribozyme of the hepatitis delta agent with eight new contacts suggested by sequence analysis of 188 cDNA clones

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