A Study on the Function of the Glycine Residue in the YGDD Motif of the RNA-Dependent RNA Polymerase β-Subunit from RNA Coliphage Qβ

Abstract

Qβreplicases in which the Gly residue of the β-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg²⁺-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Qβ phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn²⁺ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn²⁺. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Qβ replicase is involved in Mg²⁺-catalyzed polymerization. In the Mn²⁺-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.