A study on the function of the glycine residue in the YGDD motif of the RNA-dependent RNA polymerase beta-subunit from RNA coliphage Q beta 1.
Q beta replicases in which the Gly residue of the beta-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 Mg(2+)-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 Q beta 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 Q beta replicase is involved in Mg(2+)-catalyzed polymerization. In the Mn(2+)-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.
Inokuchi
Y
Y
Department of Biosciences, Teikyo University, Tochigi.
Kajitani
M
M
Hirashima
A
A
D017908:Allolevivirus / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D002384:Catalysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005998:Glycine / Q000502:physiology*; D008381:Herpesvirus 2, Gallid / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D011068:Poly G / Q000096:biosynthesis; D011777:Q beta Replicase / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D013698:Templates, Genetic
D046911:Macromolecular Substances; C028696:Qbeta ribonucleic acid; D012367:RNA, Viral; D011068:Poly G; D008345:Manganese; D011777:Q beta Replicase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium; D005998:Glycine