Substrate, metal and template effects on inhibition of bacteriophage-qβ ribonucleic acid polymerase by ortho- and pyro-phosphate

Brooks, Robert R.; Andersen, J A

doi:10.1042/bj1710725

Cited by 5 publications

(13 citation statements)

References 23 publications

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“…The ability of nucleic acid polymerases to utilize transition metals, especially Mn 2ϩ , as the divalent cation cofactor instead of Mg 2ϩ is well established (33)(34)(35)(36)(37). The primary effect of Mn 2ϩ relative to Mg 2ϩ is that nucleotide specificity is relaxed, that is nucleotides with the inappropriate sugar or base can be incorporated more efficiently (35,36).…”

Section: Dmentioning

confidence: 99%

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Arnold

Ghosh

Cameron

1999

Journal of Biological Chemistry

103

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Section: Dmentioning

confidence: 99%

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Arnold

Ghosh

Cameron

1999

Journal of Biological Chemistry

103

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“…2A, lane 4). It should be mentioned that several nucleic acid polymerases of different origins have been shown to be more efficient in the presence of Mn 2ϩ instead of Mg 2ϩ (21)(22)(23). In some cases this effect has been related to a relaxed specificity of the enzyme toward template (24) or nucleotide (22,25) over Mg 2ϩ of the poliovirus 3D pol has been attributed to a substantial reduction in the K M value of the enzyme for primer-template and to an increase in the number of the productive enzyme-primer-template complexes formed in the presence of this divalent ion (26).…”

Section: ј-Gtgaatgttatggagggcaaagcccg-3јmentioning

confidence: 99%

Identification of the Amino Acid Residue Involved in Rabbit Hemorrhagic Disease Virus VPg Uridylylation

Machin

Alonso

Parra

2001

Journal of Biological Chemistry

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The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector. The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from [␣-32 P]UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA. The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome. These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family.

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“…variety of polymerase systems, Mn 2+ has been shown to be an effective divalent cation cofactor (3)(4)(5)(6)(7). However, this metal usually alters the biochemical properties of the polymerase, decreasing the stringency of substrate selection and incorporation fidelity (4,5,(7)(8)(9)(10).…”

mentioning

confidence: 99%

Poliovirus RNA-Dependent RNA Polymerase (3D^p^o^l): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn²⁺

2004

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The use of Mn 2+ as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg 2+ often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D pol ) in the presence of Mn 2+ . The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg 2+ and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn 2+ , the conformationalchange step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn 2+ , it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotideselection process in the presence of Mg 2+ , highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn 2+ to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg 2+ , this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn 2+ may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg 2+ .All nucleic acid polymerases require two divalent cations as cofactors to catalyze phosphoryl transfer (1). The first metal ion is brought into the active site complexed to the triphosphate moiety of the nucleotide substrate. This metal ion may facilitate formation of the conformation of the triphosphate required for nucleophilic attack of the α-phosphorus atom. The second metal ion is required to lower the pK a of the primer 3′-OH to facilitate formation of the nucleophile required for catalysis. Mg 2+ is thought to be the divalent cation employed by most polymerases known (2); however, activity is usually supported by other divalent cations as well (3-7). In a Δ This work was supported, in part, by a Howard Temin Award (CA75118) from the NCI, National Institutes of Health, and by a grant (AI45818) from the NIAID, National Institute...

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Substrate, metal and template effects on inhibition of bacteriophage-qβ ribonucleic acid polymerase by ortho- and pyro-phosphate

Cited by 5 publications

References 23 publications

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Identification of the Amino Acid Residue Involved in Rabbit Hemorrhagic Disease Virus VPg Uridylylation

Poliovirus RNA-Dependent RNA Polymerase (3D^p^o^l): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn²⁺

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Substrate, metal and template effects on inhibition of bacteriophage-qβ ribonucleic acid polymerase by ortho- and pyro-phosphate

Cited by 5 publications

References 23 publications

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Poliovirus RNA-dependent RNA Polymerase (3Dpol)

Identification of the Amino Acid Residue Involved in Rabbit Hemorrhagic Disease Virus VPg Uridylylation

Poliovirus RNA-Dependent RNA Polymerase (3Dpol): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn2+

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About

Poliovirus RNA-Dependent RNA Polymerase (3D^p^o^l): Pre-Steady-State Kinetic Analysis of Ribonucleotide Incorporation in the Presence of Mn²⁺