Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme

Kumar, Penmetcha K. R.; Suh, Young‐Ah; Miyashiro, Hirotsugu; Nishikawa, Fumiko; Kawakami, Junji; Taira, Kazunari; Nishikawa, Satoshi

doi:10.1093/nar/20.15.3919

Cited by 48 publications

(43 citation statements)

References 26 publications

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“…These mutagenic results strongly support a pseudoknot-like structure for genomic HDV ribozyme [15]). Our results from an analysis of the effects of random mutations in the two single-stranded regions between positions 726 731 (SSrA region) and 762-766 (SSrB region) on the secondary structure of pseudoknot model, confirmed that these two single-stranded regions are involved in the self-cleaving activity and a few bases located in these regions may play a critical role in the catalysis [16].…”

Section: Introductionsupporting

confidence: 64%

“…The possible role of the essential nucleotides in ribozymes can be considered either to involve interactions with metal ions suggested as real catalyst recently [18,19] and/or maintenance the tertiary structure of the molecule, both of which are required for formation of the active conformation. Our previous results of internal deletion mutants [14], point and compensation mutations at stem regions [15] and random mutations [16] on HDV genomic ribozyme suggested that single-stranded regions, bases from 726-731 (SSrA) and 762-766 (SSrB), in the pseudoknot-like model (Fig. 1A), play an important role for its self-cleavage reaction.…”

Section: Discussionmentioning

confidence: 88%

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Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

Suh¹,

Kumar²,

Kawakami³

et al. 1993

FEBS Letters

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To elucidate the role of specific bases in the self‐cleavage activity of the human hepatitis delta virus (HDV) ribozyme, systematic substitutions of individual bases in two important single‐stranded regions [between nucleotides 726–731 (SSrA region) and 762–766 (SSrB region)] were carried out by oligonucleotide‐directed point mutagenesis. Among the mutants obtained, 12 mutants (G726 variants, G727A, G727C, G728C, G762A, G762C, C763 variants and A766C) could not tolerate the respective base‐substitutions and self‐cleavage activities were reduced to very low levels (10%), suggesting a requirement of the respective bases. In particular, G726 in the SSrA region and C763 in the SSrB region were found to be essential for the ribozyme activity. We could determine the preferred sequences, 5′‐G‐G‐(G/A/U)‐N‐(A/U/G)‐Pu‐3′for SSrA and 5′‐(G/U)‐C‐N‐(A/G/U)‐A‐3′ for SSrB regions, respectively.

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Section: Introductionsupporting

confidence: 64%

Section: Discussionmentioning

confidence: 88%

Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

Suh¹,

Kumar²,

Kawakami³

et al. 1993

FEBS Letters

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“…Interpretation of the mutagenesis data from different laboratories is not always consistent because different criteria for ribozyme activity are used. The faster wild-type HDV ribozyme sequences cleave with a half-life of less than 10 s (Been and Perrotta, 1995;Been et al, 1992;Thill et al, 1993), so that assays that measure the endpoint of the reaction after extensive incubation (Kawakami et al, 1993;Kumar et al, 1992Kumar et al, , 1993 only detect the slowest mutants or mutants that are misfolded into inactive structures. In an example that demonstrates extremely different assignment of significance to a particular position, a single mutation (G25-A) in the genomic ribozyme retains full activity in an endpoint assay (Kawakami et al, 1993) but is down 2.6X103-fold in rate (Tanner, 1995).…”

Section: The Self-cleavage Reactionmentioning

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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“…To elucidate the functional structure of the HDV ribozyme and to evaluate the role of bases, we have used in vitro mutagenic analyses [3][4][5] and experiments with chemical probes [6]. Our results indicate that important bases lie within three single-stranded regions (SSrA, 726-731; SSrB, 762-766; and SSrC, 708-715 in Fig.…”

Section: Introductionmentioning

confidence: 88%

“…Important bases in single-stranded regions (SSrA, B and C) of the genomic HDV ribozyme (HDV88) and antigenomic ribozyme are shown in pseudoknot secondary structure models. These data are based on our previous in vitro mutagenesis studies [3,5]. The most important residues (ones which cannot be substituted in any other bases) are shown by white letters against black squares.…”

Section: Comparison Of Trans-acting Systemsmentioning

confidence: 99%

Constructing an efficient trans‐acting genomic HDV ribozyme

Kawakami

Yuda

Suh³

et al. 1996

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We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv=0.022 min-l), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans~4 yielded kdv = 1.7 min 1. HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37°C with 1 mM of MgCl2).Key words. HDV ribozyme; Pseudoknot structure; Ribozyme, trans-acting to cleave target RNAs, cis-acting (intramolecular) ribozymes were dissected into substrates and enzymes. Earlier, efficient trans-acting hammerhead and hairpin ribozymes were constructed and showed that they function very efficiently both in vitro and in vivo [7]. Similarly, both genomic and antigenomic HDV ribozymes have been dissected into substrates and enzymes, but not yet analyzed as extensively as hammerhead and hairpin ribozymes. To increase their activity, nonphysiological conditions, such as high temperature and denaturants, have been used in reaction conditions.In the present study, we engineered the genomic HDV ribozyme to construct several trans-acting ribozymes to use in trans to cleave target RNAs. We succeeded in increasing their activity under physiological reaction conditions. Materials and methods

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Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme

Cited by 48 publications

References 26 publications

Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

Systematic substitution of individual bases in two important single‐stranded regions of the HDV ribozyme for evaluation of the role of specific bases

Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA

Constructing an efficient trans‐acting genomic HDV ribozyme

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