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The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme.

Brown AL, Perrotta AT, Wadkins TS, Been MD - Nucleic Acids Res. (2008)

Bottom Line: The cleaved fraction could be increased or decreased with mutations in the upstream sequence.Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM).Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Duke University Medical Center, Durham NC 27710, USA.

ABSTRACT
The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

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Changes in the upstream (5′) sequence can alter the extent of self-cleavage. (a) Cleavage of PAH2 (triangles) and PAH3 (diamonds) compared to the wt PAH1 (circles). The data for PAH3 (and PAH1) were collected on a rapid quench instrument. PAH2 cleavage was nearly undetectable. PAH3 cleaved to 81% with a rate constant (42 ± 2 min−1) that was similar to PAH1 (47 ± 2 min−1). Reaction conditions were 2 mM Mg2+, pH 7.5 and 25°C. (b) Cleavage of PAH4 (closed diamonds) and PAH2c (closed inverted triangles). PAH4 data collected by rapid quench generated rate constants of 41 ± 2 min−1. Data for PAH2, PAH3 and PAH2c for this experiment were collected by hand mixing, so only the extent of cleavage could be determined in those reactions.
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Figure 5: Changes in the upstream (5′) sequence can alter the extent of self-cleavage. (a) Cleavage of PAH2 (triangles) and PAH3 (diamonds) compared to the wt PAH1 (circles). The data for PAH3 (and PAH1) were collected on a rapid quench instrument. PAH2 cleavage was nearly undetectable. PAH3 cleaved to 81% with a rate constant (42 ± 2 min−1) that was similar to PAH1 (47 ± 2 min−1). Reaction conditions were 2 mM Mg2+, pH 7.5 and 25°C. (b) Cleavage of PAH4 (closed diamonds) and PAH2c (closed inverted triangles). PAH4 data collected by rapid quench generated rate constants of 41 ± 2 min−1. Data for PAH2, PAH3 and PAH2c for this experiment were collected by hand mixing, so only the extent of cleavage could be determined in those reactions.

Mentions: Introducing additional base pairs to stabilize the alternative pairing (AltP2) would be predicted to reduce the extent of antigenomic ribozyme cleavage. PAH2, the construct with a stabilized AltP2 (Figure 4a) differed from PAH1 at five positions in the upstream sequence that could form the 5′ side of AltP2 (C-19a, C-21u, U-22c, G-23u and G-25u). With the PAH2 precursor, <1% of the RNA cleaved in the initial phase of the reaction (Figure 5a and b), and no additional specific cleavage was observed with longer incubation (≤2% after 4 and 24 h at 25°C, data not shown). Considering that no mutations were made in the ribozyme domain, this result was consistent with the hypothesis that the formation of an alternate pairing (such as a stabilized AltP2) inhibited ribozyme activity in PAH2.Figure 4


The poly(A) site sequence in HDV RNA alters both extent and rate of self-cleavage of the antigenomic ribozyme.

Brown AL, Perrotta AT, Wadkins TS, Been MD - Nucleic Acids Res. (2008)

Changes in the upstream (5′) sequence can alter the extent of self-cleavage. (a) Cleavage of PAH2 (triangles) and PAH3 (diamonds) compared to the wt PAH1 (circles). The data for PAH3 (and PAH1) were collected on a rapid quench instrument. PAH2 cleavage was nearly undetectable. PAH3 cleaved to 81% with a rate constant (42 ± 2 min−1) that was similar to PAH1 (47 ± 2 min−1). Reaction conditions were 2 mM Mg2+, pH 7.5 and 25°C. (b) Cleavage of PAH4 (closed diamonds) and PAH2c (closed inverted triangles). PAH4 data collected by rapid quench generated rate constants of 41 ± 2 min−1. Data for PAH2, PAH3 and PAH2c for this experiment were collected by hand mixing, so only the extent of cleavage could be determined in those reactions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 5: Changes in the upstream (5′) sequence can alter the extent of self-cleavage. (a) Cleavage of PAH2 (triangles) and PAH3 (diamonds) compared to the wt PAH1 (circles). The data for PAH3 (and PAH1) were collected on a rapid quench instrument. PAH2 cleavage was nearly undetectable. PAH3 cleaved to 81% with a rate constant (42 ± 2 min−1) that was similar to PAH1 (47 ± 2 min−1). Reaction conditions were 2 mM Mg2+, pH 7.5 and 25°C. (b) Cleavage of PAH4 (closed diamonds) and PAH2c (closed inverted triangles). PAH4 data collected by rapid quench generated rate constants of 41 ± 2 min−1. Data for PAH2, PAH3 and PAH2c for this experiment were collected by hand mixing, so only the extent of cleavage could be determined in those reactions.
Mentions: Introducing additional base pairs to stabilize the alternative pairing (AltP2) would be predicted to reduce the extent of antigenomic ribozyme cleavage. PAH2, the construct with a stabilized AltP2 (Figure 4a) differed from PAH1 at five positions in the upstream sequence that could form the 5′ side of AltP2 (C-19a, C-21u, U-22c, G-23u and G-25u). With the PAH2 precursor, <1% of the RNA cleaved in the initial phase of the reaction (Figure 5a and b), and no additional specific cleavage was observed with longer incubation (≤2% after 4 and 24 h at 25°C, data not shown). Considering that no mutations were made in the ribozyme domain, this result was consistent with the hypothesis that the formation of an alternate pairing (such as a stabilized AltP2) inhibited ribozyme activity in PAH2.Figure 4

Bottom Line: The cleaved fraction could be increased or decreased with mutations in the upstream sequence.Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM).Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Duke University Medical Center, Durham NC 27710, USA.

ABSTRACT
The ribozyme self-cleavage site in the antigenomic sequence of hepatitis delta virus (HDV) RNA is 33-nt downstream of the poly(A) site for the delta antigen mRNA. An HDV antigenomic ribozyme precursor RNA that included the upstream poly(A) processing site was used to test the hypothesis that nonribozyme sequence near the poly(A) site could affect ribozyme activity. Relative to ribozyme precursor without the extra upstream sequences, the kinetic profile for self-cleavage of the longer precursor was altered in two ways. First, only half of the precursor RNA self-cleaved. The cleaved fraction could be increased or decreased with mutations in the upstream sequence. These mutations, which were predicted to alter the relative stability of competing secondary structures within the precursor, changed the distribution of alternative RNA structures that are resolved in native-gel electrophoresis. Second, the active fraction cleaved with an observed rate constant that was higher than that of the ribozyme without the upstream sequences. Moreover, the higher rate constants occurred at lower, near-physiological, divalent metal ion concentrations (1-2 mM). Modulation of ribozyme activity, through competing alternative structures, could be part of a mechanism that allows a biologically significant choice between maturation of the mRNA and processing of replication intermediates.

Show MeSH
Related in: MedlinePlus