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

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.