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Related Experiment Videos

Nucleotide binding by the poliovirus RNA polymerase.

O C Richards1, P Yu, K L Neufeld

  • 1Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.

The Journal of Biological Chemistry
|August 25, 1992
PubMed
Summary
This summary is machine-generated.

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Poliovirus RNA polymerase binds nucleotides at a single site, identified via cross-linking studies. This binding stabilizes the enzyme, aiding purification and revealing mutation effects on nucleotide interaction.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • Poliovirus RNA-dependent RNA polymerase is crucial for viral replication.
  • Understanding nucleotide binding is key to enzyme function and inhibition.
  • Previous studies lacked detailed characterization of the NTP binding site.

Purpose of the Study:

  • To characterize the nucleotide binding site on poliovirus RNA polymerase.
  • To investigate the effect of nucleotide binding on enzyme stability and conformation.
  • To explore the utility of nucleotide binding for enzyme purification and analysis.

Main Methods:

  • Cross-linking of ribonucleoside triphosphates (NTPs) to the polymerase using UV irradiation and chemical reduction.
  • Peptide mapping and sequencing of cross-linked enzyme fragments.

Related Experiment Videos

  • GTP-agarose affinity chromatography for enzyme purification and analysis of mutant polymerases.
  • Thermal denaturation assays to assess enzyme stability.

    • Main Results:

    • A single binding site for all NTPs was identified on the poliovirus RNA polymerase.
    • Cross-linking occurred to a lysine residue between Met-189 and Lys-228.
    • GTP-agarose affinity chromatography enabled efficient enzyme purification.
    • Mutations affected GTP binding affinity, with some eliminating it entirely.
    • NTP binding, especially GTP, significantly protected the polymerase against heat denaturation and activity loss.

    Conclusions:

    • Nucleotide binding induces a conformational change or stabilizes an ordered conformation of the poliovirus RNA polymerase.
    • The identified binding site and its characteristics are critical for polymerase function.
    • Nucleotide binding properties can be leveraged for enzyme purification and studying structure-function relationships.