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Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.

Saran D, Held DM, Burke DH - Nucleic Acids Res. (2006)

Bottom Line: The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions.By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1).The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.

ABSTRACT
Ribozymes that phosphorylate internal 2'-OH positions mimic the first mechanistic step of P-type ATPase enzymes by forming a phospho-enzyme intermediate. We previously described 2'-autophosphorylation and autothiophosphorylation by the 2PTmin3.2 ribozyme. In the present work we demonstrate that the thiophosphorylated form of this ribozyme can de-thiophosphorylate in the absence of ATPgammaS. Identical ionic conditions yield a thiophosphorylated strand when ATPgammaS is included, thus effecting a net ATPgammaS hydrolysis. The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions. By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1). The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

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Related in: MedlinePlus

De-thiophosphorylation. (A) The percent of DRD26 in the (thio)phosphorylated form (i.e. unreacted) is plotted as a function of time for substrate strand incubated in the presence of ribozyme 2PTmin3.2 and ADP (diamonds), ribozyme only (circles), or by itself (triangles). Asterisk represents the de-thiophosphorylation of substrate strand DRD26 that had been auto-phosphorylated at G10S by [32P]ATP. (B) and (C) Autoradiographs of typical gels showing loss of thiophosphoryl group from end-labeled RNA in the absence (B) or presence (C) of the reducing agent tris-carboxyethylphosphine (TCEP) (13). De-thiophosphorylation times in hrs are indicated above each lane. A weak oxidized dimer product just above the de-thiophosphorylated monomer is evident at long times when TCEP is omitted. Black bars indicate locations and thicknesses of APM layers. (D) Predicted structure of potential dimer of the substrate strand. (E) Concentration dependence. De-thiophosphorylation of 0.5 μM substrate strand was monitored in the presence of 0 (diamonds), 0.5 μM (squares), 1 μM (circles), 1.5 μM (asterisks) and 2 μM (solid triangles) unlabeled substrate strand DRD26. De-thiophosphorylation of 2.5 μM substrate strand was also monitored in the presence of 10 μM ribozyme strand (open triangles).
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fig2: De-thiophosphorylation. (A) The percent of DRD26 in the (thio)phosphorylated form (i.e. unreacted) is plotted as a function of time for substrate strand incubated in the presence of ribozyme 2PTmin3.2 and ADP (diamonds), ribozyme only (circles), or by itself (triangles). Asterisk represents the de-thiophosphorylation of substrate strand DRD26 that had been auto-phosphorylated at G10S by [32P]ATP. (B) and (C) Autoradiographs of typical gels showing loss of thiophosphoryl group from end-labeled RNA in the absence (B) or presence (C) of the reducing agent tris-carboxyethylphosphine (TCEP) (13). De-thiophosphorylation times in hrs are indicated above each lane. A weak oxidized dimer product just above the de-thiophosphorylated monomer is evident at long times when TCEP is omitted. Black bars indicate locations and thicknesses of APM layers. (D) Predicted structure of potential dimer of the substrate strand. (E) Concentration dependence. De-thiophosphorylation of 0.5 μM substrate strand was monitored in the presence of 0 (diamonds), 0.5 μM (squares), 1 μM (circles), 1.5 μM (asterisks) and 2 μM (solid triangles) unlabeled substrate strand DRD26. De-thiophosphorylation of 2.5 μM substrate strand was also monitored in the presence of 10 μM ribozyme strand (open triangles).

Mentions: When ribozyme 2PTmin3.2 is annealed with 5′ radiolabeled substrate strand DRD26 and incubated in 5 mM ATPγS, thiophosphorylation of the substrate proceeds with a first-order rate constant, kobs ≈ 0.21 h−1, similar to values obtained previously (12). Thiophosphorylated DRD26 was purified from the organomercurial layer of a trilayer gel containing acryloylamino-phenylmercuric chloride (APM) (13,14), annealed to ribozyme strand, and incubated in selection buffer in the presence of 5 mM ADP. When the products of this reaction were separated on a second trilayer organomercurial gel, the fraction of the radioactivity that shifted into the APM layer decreased with time, indicating loss of thiophosphate from the radiolabeled strand (Figure 2A, diamonds, and Figure 2B). Interestingly, there is a highly reproducible lag in the reaction, with little de-thiophosphorylation occurring within the first hour followed by an increased rate of loss of thiophosphate. Similar biphasic kinetics were observed throughout this study.


Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.

Saran D, Held DM, Burke DH - Nucleic Acids Res. (2006)

De-thiophosphorylation. (A) The percent of DRD26 in the (thio)phosphorylated form (i.e. unreacted) is plotted as a function of time for substrate strand incubated in the presence of ribozyme 2PTmin3.2 and ADP (diamonds), ribozyme only (circles), or by itself (triangles). Asterisk represents the de-thiophosphorylation of substrate strand DRD26 that had been auto-phosphorylated at G10S by [32P]ATP. (B) and (C) Autoradiographs of typical gels showing loss of thiophosphoryl group from end-labeled RNA in the absence (B) or presence (C) of the reducing agent tris-carboxyethylphosphine (TCEP) (13). De-thiophosphorylation times in hrs are indicated above each lane. A weak oxidized dimer product just above the de-thiophosphorylated monomer is evident at long times when TCEP is omitted. Black bars indicate locations and thicknesses of APM layers. (D) Predicted structure of potential dimer of the substrate strand. (E) Concentration dependence. De-thiophosphorylation of 0.5 μM substrate strand was monitored in the presence of 0 (diamonds), 0.5 μM (squares), 1 μM (circles), 1.5 μM (asterisks) and 2 μM (solid triangles) unlabeled substrate strand DRD26. De-thiophosphorylation of 2.5 μM substrate strand was also monitored in the presence of 10 μM ribozyme strand (open triangles).
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Related In: Results  -  Collection

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fig2: De-thiophosphorylation. (A) The percent of DRD26 in the (thio)phosphorylated form (i.e. unreacted) is plotted as a function of time for substrate strand incubated in the presence of ribozyme 2PTmin3.2 and ADP (diamonds), ribozyme only (circles), or by itself (triangles). Asterisk represents the de-thiophosphorylation of substrate strand DRD26 that had been auto-phosphorylated at G10S by [32P]ATP. (B) and (C) Autoradiographs of typical gels showing loss of thiophosphoryl group from end-labeled RNA in the absence (B) or presence (C) of the reducing agent tris-carboxyethylphosphine (TCEP) (13). De-thiophosphorylation times in hrs are indicated above each lane. A weak oxidized dimer product just above the de-thiophosphorylated monomer is evident at long times when TCEP is omitted. Black bars indicate locations and thicknesses of APM layers. (D) Predicted structure of potential dimer of the substrate strand. (E) Concentration dependence. De-thiophosphorylation of 0.5 μM substrate strand was monitored in the presence of 0 (diamonds), 0.5 μM (squares), 1 μM (circles), 1.5 μM (asterisks) and 2 μM (solid triangles) unlabeled substrate strand DRD26. De-thiophosphorylation of 2.5 μM substrate strand was also monitored in the presence of 10 μM ribozyme strand (open triangles).
Mentions: When ribozyme 2PTmin3.2 is annealed with 5′ radiolabeled substrate strand DRD26 and incubated in 5 mM ATPγS, thiophosphorylation of the substrate proceeds with a first-order rate constant, kobs ≈ 0.21 h−1, similar to values obtained previously (12). Thiophosphorylated DRD26 was purified from the organomercurial layer of a trilayer gel containing acryloylamino-phenylmercuric chloride (APM) (13,14), annealed to ribozyme strand, and incubated in selection buffer in the presence of 5 mM ADP. When the products of this reaction were separated on a second trilayer organomercurial gel, the fraction of the radioactivity that shifted into the APM layer decreased with time, indicating loss of thiophosphate from the radiolabeled strand (Figure 2A, diamonds, and Figure 2B). Interestingly, there is a highly reproducible lag in the reaction, with little de-thiophosphorylation occurring within the first hour followed by an increased rate of loss of thiophosphate. Similar biphasic kinetics were observed throughout this study.

Bottom Line: The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions.By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1).The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.

ABSTRACT
Ribozymes that phosphorylate internal 2'-OH positions mimic the first mechanistic step of P-type ATPase enzymes by forming a phospho-enzyme intermediate. We previously described 2'-autophosphorylation and autothiophosphorylation by the 2PTmin3.2 ribozyme. In the present work we demonstrate that the thiophosphorylated form of this ribozyme can de-thiophosphorylate in the absence of ATPgammaS. Identical ionic conditions yield a thiophosphorylated strand when ATPgammaS is included, thus effecting a net ATPgammaS hydrolysis. The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions. By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1). The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

Show MeSH
Related in: MedlinePlus