Probing the legitimate initiation of RNA synthesis by Qβ replicase with oligonucleotide primers

• Published in: FEBS letters 2024-03, Vol.598 (5), p.579-586
• Main Authors: Lobodin, Kirill V., Chetverina, Helena V., Chetverin, Alexander B.
• Format: Article
• Language: English
• Subjects: 5′‐triphosphate; elongation factor EF‐Tu; RNA secondary structure; RNA‐directed RNA polymerase; single‐stranded RNA phage; template recognition
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1002/1873-3468.14833

Oligoribonucleotides complementary to the template 3′ terminus were tested for their ability to initiate RNA synthesis on legitimate templates capable of exponential amplification by Qβ replicase. Oligonucleotides shorter than the distance to the nearest predicted template hairpin proved able to serve as primers, with the optimal length varying for different templates, suggesting that during initiation the template retains its native fold incorporating the 3′ terminus. The priming activity of an oligonucleotide is greatly enhanced by its 5′‐triphosphate group, the effect being strongly dependent on Mg2+ ions. This indicates that, unlike other studied RNA polymerases, Qβ replicase binds the 5′‐triphosphate of the initiating nucleotide GTP, and this binding is needed for the replication of legitimate templates. An oligonucleotide shorter than the distance to the nearest template hairpin can initiate RNA synthesis on a legitimate Qβ replicase template. The optimal primer length varies for different templates. The primer binding is greatly stabilized by its 5′‐triphosphate group and Mg2+ ions.