Function and structure in ribonucleic acid phage Qbeta ribonucleic acid replicase. Effect of inhibitors of EF-Tu on ribonucleic acid synthesis and renaturation of active enzyme.
Escherichia coli Phage Qbeta RNA replicase, an RNA-dependent RNA polymerase, 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, are required for initiation of transcription by Qbeta replicase with all templates. Using a previously developed reconstitution system we have examined the effects of modification of EF-Tu on reconstituted replicase activity. The poly(G) polymerase activity of the enzyme can be recovered after pretreatment of the EF-Tu-GDP with either L-1-tosylamido-2-phenylethyl chloromethyl ketone or N-ethylmaleimide, both of which inhibit the aminoacyl-tRNA binding activity of EF-Tu. This suggests that the aminoacyl-tRNA binding site of EF-Tu is not required for Qbeta replicase activity. When Qbeta replicase is treated with kirromycin, an antibiotic which modifies EF-Tu activity by an unknown mechamism, the protein synthetic activity of the EF-Tu in the replicase complex is eliminated but the Qbeta RNA replication activity is only slightly affected. Treatment of pure EF-Tu with kirromycin, however, prevents it from functioning in the renaturation of Qbeta replicase. This antibiotic is not effective against the EF-Tu-Ts complex in the reconstitution assay. Kirromycin at the relatively high concentration used here is found to prevent the formation of the EF-Tu-Ts complex. GDP, which binds to EF-Tu and inhibits formation of the complex with EF-Ts, also inhibits renaturation of Qbeta replicase. It is suggested that the EF-Tu-Ts complex, rather than the individual polypeptides, functions in the renaturation of Qbeta replicase and that the kirromycin and GDP act by preventing formation of this complex.
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001665:Binding Sites; D005033:Ethylmaleimide / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D010445:Peptide Elongation Factors; D011485:Protein Binding; D011489:Protein Denaturation; D011777:Q beta Replicase / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D014105:Tosyl Compounds / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D014105:Tosyl Compounds; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases; D005033:Ethylmaleimide