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Probing the nucleobase selectivity of RNA polymerases with dual-coding substrates

Janne J Mäkinen1, Petja Rosenqvist2, Pasi Virta2

  • 1Department of Life Technologies, University of Turku, Turku, Finland.

The Journal of Biological Chemistry
|September 11, 2024
PubMed

Insights

Formycin A and pyrazofurin A are nucleoside analogs that can be incorporated by RNA polymerases. These analogs exhibit unique base-pairing abilities, offering potential for new antiviral therapies.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Virology

Background:

  • Nucleoside analogs like Formycin A (FOR) and pyrazofurin A (PYR) possess antiviral and antitumor activities.
  • Their precise mechanisms impacting nucleic acid metabolism and transcription remain incompletely understood.

Purpose of the Study:

  • To investigate the utilization of FOR, PYR, and oxidized purine nucleotides by bacterial, mitochondrial, and viral RNA polymerases (RNAPs).
  • To elucidate the substrate incorporation and base-pairing specificities of structurally diverse RNAPs.

Main Methods:

  • Enzymatic assays to assess the incorporation of FOR, PYR, and oxidized purines by various RNAPs.
  • Analysis of base-pairing interactions and geometric compatibility during nucleotide incorporation.

Main Results:

  • All tested RNAPs incorporated FOR in place of adenine and PYR in place of uridine.
  • FOR demonstrated dual-coding, substituting for cytosine, especially in viral RNAP.
  • Oxidized purines (8-oxoadenine, 8-oxoguanine) were incorporated as uridine analogs alongside canonical pairings.
  • RNAP structural diversity influenced the utilization of alternative nucleotide conformers (syn).

Conclusions:

  • The ability of FOR and oxidized purines to adopt alternative conformers underlies their dual-coding potential.
  • RNAP's nucleobase selection mechanisms vary based on enzyme structure and substrate conformation.
  • These findings provide novel insights into RNAP function and could inform the development of advanced antiviral therapies.
Keywords:
8-oxoadenine8-oxoguanineRNA polymerasebacterial transcriptionenzyme kineticsformycinnucleoside analogribavirinsubstrate specificityyprazofurin

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