Reconstitution of Qβ RNA replicase from a covalently bonded elongation factor Tu.Ts complex

Abstract

Escherichia coli phage Qβ RNA replicase, an RNA dependent RNA polymerase (RNA dependent RNA nucleotidyltransferase), is a tetramer composed of one phage coded polypeptide and three host supplied polypeptides which are known to function in the biosynthesis of proteins in the uninfected host. Two of these polypeptides, protein synthesis elongation factors EF Tu and EF Ts, can be covalently crosslinked with dimethyl suberimidate to form a complex which lacks the ability to catalyze the known host functions catalyzed by the individual elongation factors. Using a previously developed reconstitution system the authors have examined the effects of crosslinking the EF Tu.Ts complex on reconstituted replicase activity. Renaturation is significantly more efficient when exogenously added native EF Tu.Ts is crosslinked than when it is not. Crosslinked EF Tu.Ts can be purified from a crude crosslinked postribosomal supernatant by its ability to replace EF Tu and Ef Ts in the renaturation of denatured Qβ replicase. A sample of Qβ replicase with crosslinked EF Tu.Ts replacing the individual elongation factors was prepared. Although it lacked EF Tu and Ef Ts activities, it could initiate transcription of both poly(C) and Qβ RNA normally and had approximately the same specific activity as control enzyme. Denatured Qβ replicase formed with crosslinked EF Tu.Ts was found to renature much more rapidly than untreated enzyme and, in contrast to normal replicase, its renaturation was not inhibited by GDP. The results demonstrate that EF Tu and EF Ts function as a complex in Qβ replicase and do not perform their known protein biosynthetic functions in the RNA synthetic reaction.