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

Abstract

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.

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

“…Here, the priming activity of GTP is not to be compared with that of oligonucleotides since GTP is present at a 10‐fold higher concentration (cf. Materials and methods) and is converted into a mixture of oligoG during the initiation step [7].…”

“…RNA templates, the (+) and (−) strands of RQ87 −3 RNA [7] and RQ120 −2 RNA (a subspecies of RQ120 RNA [11], Fig. S1), were prepared by run‐off transcription with T7 RNA polymerase [7]. The reaction temperature was 22 °C.…”

“…The reaction temperature was 22 °C. For initiation, 0.5 pmol of a template was incubated for 10 min with 5 pmol of the single‐chain Qβ replicase core [7,12] in 8 μL of a 1.25× buffer Q (50 m m Tris–HCl, pH 7.5 at 25 °C, 10 m m MgCl 2 , 1 m m EDTA) in the presence of 125 μ m GTP or 12.5 μ m oligonucleotide as indicated, followed by the addition of 1 μL of 10 m m aurintricarboxylic acid. A 10‐min elongation was started 1 min later by the addition of 1 μL of a mixture of rNTPs to the final concentrations of 1 m m GTP, ATP, and CTP and 0.01 m m α 32 P‐UTP (0.1–1 MBq·mL −1 ).…”

“…A 10‐min elongation was started 1 min later by the addition of 1 μL of a mixture of rNTPs to the final concentrations of 1 m m GTP, ATP, and CTP and 0.01 m m α 32 P‐UTP (0.1–1 MBq·mL −1 ). The labeled reaction products were analyzed using non‐denaturing gel electrophoresis and visualized by autoradiography as reported [7]. The size marker for a single‐stranded reaction product was synthesized with T7 RNA polymerase in the presence of α 32 P‐UTP, and that for a double‐stranded product was prepared by annealing the labeled transcript with an excess of the complementary unlabeled transcript, by heating at 99° for 2 min in 100 m m Tris–НСl, pH 8.0, 500 m m NaCl, 1 m m EDTA, followed by slow cooling to room temperature.…”

“…The ATA‐resistant replicase–template complex is unstable, but is stabilized in the presence of the initiator nucleotide GTP due to the synthesis of oligoG complementary to the template 3′‐terminal oligoC [3]; however, even then it decomposes within several minutes, and only at the elongation phase does the transition to the closed conformation become nearly irreversible [6]. Therefore, continuous oligoG synthesis occurs when the replicase is incubated with a legitimate template and GTP, resulting in a heterogenous population of initiation complexes containing GTP or oligoG of different lengths, including long strands produced due to transcript slippage [7]. The metastability and heterogeneity of the legitimate initiation complexes hinder their studies.…”