Function and Structure in Ribonucleic Acid Phage Qβ Ribonucleic Acid Replicase

Phage Qβ RNA-dependent RNA replicase consists of four subunits: one is RNA phage specific (subunit II), the other three, present in the uninfected cell, are the protein biosynthesis elongation factors EF-Tu (subunit III) and EF-Ts (subunit IV) and the translation interference factor i (subunit I). When the protein biosynthesis elongation factors are removed by centrifugation of the initiated enzyme-RNA complex, the remaining two subunits (I and II) are capable of continued polymerization at a normal rate but do not initiate subsequent rounds of synthesis. Thus the protein biosynthesis factors are required for initiation but not elongation of RNA synthesis by Qβ replicase. Similar results are observed when this experiment is performed with enzyme lacking subunit I, indicating that the RNA polymerizing activity of the enzyme resides in the phage-coded polypeptide, subunit II. Guanosine tetraphosphate, a strong competitor of GTP binding by EF-Tu, inhibits initiation but not elongation by Qβ replicase on synthetic templates. However, the role of EF-T in the initiation of RNA synthesis on synthetic templates is not simply related to the functions it performs in protein biosynthesis. The aminoacyl-tRNA binding activity of EF Tu in the Qβ replicase complex can be inactivated by alkylating reagents without affecting replicase poly(U, G) polymerase activity. 5′-Guanylyl-β, γ-imidodiphosphate, an inhibitor of EF-Tu-catalyzed protein biosynthesis due to its resistance to cleavage to GDP, does not inhibit replicase activity although the compound is a competitor of GDP binding. The protein biosynthesis factors appear to play an essential role in maintaining the active conformation of the replicase enzyme complex. Catalytic activities of both EF-Tu and EFTs can be assayed when the factors are part of Qβ replicase, but the activities differ from those of the isolated factors in their sensitivity to ionic strength and dissociation by GDP.