Phage Qbeta replicase: cell-free synthesis of the phage-specific subunit and its assembly with host subunits to form active enzyme.
Cell-free translation of Qbeta RNA and subsequent partial purification of the enzyme resulted in replicase activity. From 0.5 to 1.5% of all R chains synthesised were found in the 7-S replicase complex. The presence in the 7-S complex of the host subunits of authentic replicase, i (= S1) and EF-Ts, was shown by the effect of antisera directed against ribosomal protein S1 and EF-Ts, respectively. Furthermore, the presence of EF-Ts was demonstrated by thermal denaturation of in vitro replicase made by a cell extract from an Escherichia coli mutant with a thermolabile EF-Ts. In vitro replicase did not assemble spontaneously during protein synthesis but was formed upon subsequent purification. Assembly could be induced by ammonium sulphate precipitation (60% saturation) alone. It is concluded that the functional phage-coded subunit synthesised in vitro recognises i and the EF-Tu - EF-Ts complex among a mixture of host proteins.
D000645:Ammonium Sulfate; D002474:Cell-Free System; D003090:Coliphages / Q000201:enzymology*; D004926:Escherichia coli / Q000276:immunology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D009154:Mutation; D010445:Peptide Elongation Factors / Q000032:analysis; D011777:Q beta Replicase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism
D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D012367:RNA, Viral; D006639:Histidine; D006160:Guanosine Triphosphate; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases; D000645:Ammonium Sulfate