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A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

Kolev NG, Hartland EI, Huber PW - Nucleic Acids Res. (2008)

Bottom Line: The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage.Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts.Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

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Kinetics of the cleavage reaction. (A) A trace amount of internally labeled Vg1 RNA was added to the indicated concentration of unlabeled Vg1 RNA. The reactions proceeded in standard conditions overnight. (B) Vg1 RNA (100 nM) was incubated for the indicated amount of time in standard conditions. Reactions were stopped with EDTA and analyzed on a denaturing polyacrylamide gel. (C) The autoradiograph was scanned with a laser densitometer to generate the individual data points. The best-fit curve (solid line) is defined by a kinetic equation that represents a two-step process with both steps being first order. (D) The plot of ln(fraction unreacted) versus time yields an observed first-order rate constant of 6.6 × 10−4 min−1.
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Figure 3: Kinetics of the cleavage reaction. (A) A trace amount of internally labeled Vg1 RNA was added to the indicated concentration of unlabeled Vg1 RNA. The reactions proceeded in standard conditions overnight. (B) Vg1 RNA (100 nM) was incubated for the indicated amount of time in standard conditions. Reactions were stopped with EDTA and analyzed on a denaturing polyacrylamide gel. (C) The autoradiograph was scanned with a laser densitometer to generate the individual data points. The best-fit curve (solid line) is defined by a kinetic equation that represents a two-step process with both steps being first order. (D) The plot of ln(fraction unreacted) versus time yields an observed first-order rate constant of 6.6 × 10−4 min−1.

Mentions: The extent of Vg1 mRNA cleavage exhibits little dependence on RNA concentration (Figure 3A), indicating that the reaction is first order. Decreased activity at high concentrations of the RNA actually suggests that intermolecular interactions may block or hinder formation of the active conformation in the RNA needed for catalysis. The time course of the cleavage reaction not only provides further evidence for a first-order reaction, but also that it is a two-step kinetic process (Figure 3B–D). Reaction products were separated on a denaturing polyacrylamide gel and the resulting autoradiograph was scanned with a laser densitometer in order to quantitate the intensity of the individual bands. The progress curve (Figure 3C) is best fit to a rate expression that represents a two-step mechanism in which both steps are first order. This two-step mechanism suggests that a conformational change in the RNA to a catalytically competent structure may be partially rate limiting. Biphasic kinetic behavior, also ascribed to an equilibrium between conformational states, has been reported for group I (11) and group II (12,13) self-splicing introns. The plot of ln(fraction unreacted) versus time (Figure 3D) yielded an observed first-order rate constant of 6.6 × 10–4 min−1 which is similar to rate constants measured for naturally occurring and model manganese-dependent GAAA ribozymes (14,15). This slow reaction rate indicates that the catalytically competent conformation of the ribozyme may not be highly populated in the free RNA.Figure 3.


A manganese-dependent ribozyme in the 3'-untranslated region of Xenopus Vg1 mRNA.

Kolev NG, Hartland EI, Huber PW - Nucleic Acids Res. (2008)

Kinetics of the cleavage reaction. (A) A trace amount of internally labeled Vg1 RNA was added to the indicated concentration of unlabeled Vg1 RNA. The reactions proceeded in standard conditions overnight. (B) Vg1 RNA (100 nM) was incubated for the indicated amount of time in standard conditions. Reactions were stopped with EDTA and analyzed on a denaturing polyacrylamide gel. (C) The autoradiograph was scanned with a laser densitometer to generate the individual data points. The best-fit curve (solid line) is defined by a kinetic equation that represents a two-step process with both steps being first order. (D) The plot of ln(fraction unreacted) versus time yields an observed first-order rate constant of 6.6 × 10−4 min−1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553595&req=5

Figure 3: Kinetics of the cleavage reaction. (A) A trace amount of internally labeled Vg1 RNA was added to the indicated concentration of unlabeled Vg1 RNA. The reactions proceeded in standard conditions overnight. (B) Vg1 RNA (100 nM) was incubated for the indicated amount of time in standard conditions. Reactions were stopped with EDTA and analyzed on a denaturing polyacrylamide gel. (C) The autoradiograph was scanned with a laser densitometer to generate the individual data points. The best-fit curve (solid line) is defined by a kinetic equation that represents a two-step process with both steps being first order. (D) The plot of ln(fraction unreacted) versus time yields an observed first-order rate constant of 6.6 × 10−4 min−1.
Mentions: The extent of Vg1 mRNA cleavage exhibits little dependence on RNA concentration (Figure 3A), indicating that the reaction is first order. Decreased activity at high concentrations of the RNA actually suggests that intermolecular interactions may block or hinder formation of the active conformation in the RNA needed for catalysis. The time course of the cleavage reaction not only provides further evidence for a first-order reaction, but also that it is a two-step kinetic process (Figure 3B–D). Reaction products were separated on a denaturing polyacrylamide gel and the resulting autoradiograph was scanned with a laser densitometer in order to quantitate the intensity of the individual bands. The progress curve (Figure 3C) is best fit to a rate expression that represents a two-step mechanism in which both steps are first order. This two-step mechanism suggests that a conformational change in the RNA to a catalytically competent structure may be partially rate limiting. Biphasic kinetic behavior, also ascribed to an equilibrium between conformational states, has been reported for group I (11) and group II (12,13) self-splicing introns. The plot of ln(fraction unreacted) versus time (Figure 3D) yielded an observed first-order rate constant of 6.6 × 10–4 min−1 which is similar to rate constants measured for naturally occurring and model manganese-dependent GAAA ribozymes (14,15). This slow reaction rate indicates that the catalytically competent conformation of the ribozyme may not be highly populated in the free RNA.Figure 3.

Bottom Line: The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage.Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts.Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.

ABSTRACT
The smallest catalytic RNA identified to date is a manganese-dependent ribozyme that requires only a complex between GAAA and UUU to effect site-specific cleavage. We show here that this ribozyme occurs naturally in the 3'-UTR of Vg1 and beta-actin mRNAs. In accord with earlier studies with model RNAs, cleavage occurs only in the presence of manganese or cadmium ions and proceeds optimally near 30 degrees C and physiological pH. The time course of cleavage in Vg1 mRNA best fits a two-step process in which both steps are first-order. In Vg1 mRNA, the ribozyme is positioned adjacent to a polyadenylation signal, but has no influence on translation of the mRNA in Xenopus oocytes. Putative GAAA ribozyme structures are also near polyadenylation sites in yeast and rat actin mRNAs. Analysis of sequences in the PolyA Cleavage Site and 3'-UTR Database (PACdb) revealed no particular bias in the frequency or distribution of the GAAA motif that would suggest that this ribozyme is currently or was recently used for cleavage to generate processed transcripts. Nonetheless, we speculate that the complementary strands that comprise the ribozyme may account for the origin of sequence elements that direct present-day 3'-end processing of eukaryotic mRNAs.

Show MeSH
Related in: MedlinePlus