Function and structure in ribonucleic acid phage Q beta ribonucleic acid replicase. The roles of the different subunits in transcription of synthetic templates.

Phage Q/3 RNA-dependent RNA replicate 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 Q/3 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 Q/3 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 QP replicase complex can be inactivated by alkylating reagents without affecting replicase poly(U, G) polymerase activity. 5’-Guanylyl-/I, y-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 Q/l replicase,