Slippage at the initiation of RNA synthesis by Qβ replicase results in a periodic polyG pattern

• Published in: FEBS letters 2023-02, Vol.597 (3), p.458-471
• Main Authors: Lobodin, Kirill V., Chetverina, Helena V., Chetverin, Alexander B.
• Format: Article
• Language: English
• Subjects: Guanosine Triphosphate - metabolism; Hoogsteen interactions; Protein Binding; Q beta Replicase - chemistry; Q beta Replicase - genetics; Q beta Replicase - metabolism; ribosomal protein S1; RNA - metabolism; RNA quadruplex; RNA, Viral - chemistry; RNA, Viral - genetics; RNA-Dependent RNA Polymerase; RNA‐directed RNA polymerase; single‐stranded RNA phage; transcriptional slippage
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1002/1873-3468.14556

The repetitive copying of template nucleotides due to transcriptional slippage has not been reported for RNA‐directed RNA polymerases of positive‐strand RNA phages. We unexpectedly observed that, with GTP as the only substrate, Qβ replicase, the RNA‐directed RNA polymerase of bacteriophage Qβ, synthesizes by transcriptional slippage polyG strands, which on denaturing electrophoresis produce a ladder with at least three clusters of bolder bands. The ≈ 15‐nt‐long G15, the major product of the shortest cluster, is tightly bound by the enzyme but can be released by the ribosomal protein S1, which, as a Qβ replicase subunit, normally promotes the release of a completed transcript. 7‐deaza‐GTP suppresses the polyG synthesis and abolishes the periodic pattern, suggesting that the N7 atom is needed for the initiation of RNA synthesis and the formation of the structure recognized by protein S1. The results provide new insights into the mechanism of RNA synthesis by the RNA‐directed RNA polymerase of a single‐stranded RNA phage. With GTP as the only substrate, Qβ replicase synthesizes long polyG strands on a replicable template ending with …CCC. Strands of approximately 15, 25 and 35 nt are produced in greater amounts. Ribosomal protein S1, serving as a termination factor for the replicase, recognizes the 15‐nt‐long product as a transcription termination signal.