Transfer RNA cross-linked to the elongation factor Tu subunit of Q beta replicase does not inhibit Q beta RNA replication.
One of the four subunits of bacteriophage Q beta RNA replicase is elongation factor Tu (EF-Tu), the host aminoacyl-tRNA (AA-tRNA) binding protein. To determine whether the RNA polymerase activity requires the tRNA binding site of EF-Tu, we reconstituted replicase with EF-Tu . GTP covalently bound to AA-tRNA. This cross-linked ternary complex (XLTC) was formed by the reaction of N epsilon-bromoacetyl-Lys-tRNA with EF-Tu-GTP. In an EF-Tu-dependent system for the reconstitution of replicase, XLTC restored polymerase activity at least as well as an equivalent amount of EF-Tu. Replicase reconstituted with XLTC was resolved from replicase containing EF-Tu by chromatography on phosphocellulose, a result which confirmed that the tRNA moiety was incorporated into the enzyme. Chromatographic analysis of reconstitution mixtures revealed that XLTC was incorporated into replicase as extensively as EF-Tu. From these results, it appears that the AA-tRNA binding site on EF-Tu is not required for the assembly or activity of Q beta RNA replicase. Furthermore, because the tRNA macromolecule is cross-linked to His-66 of the EF-Tu, the region surrounding His-66 must normally be exposed on the surface of the replicase.
D001426:Bacterial Proteins / Q000378:metabolism*; D003090:Coliphages / Q000378:metabolism*; D003432:Cross-Linking Reagents; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D009695:Nucleic Acid Renaturation; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011068:Poly G; D011777:Q beta Replicase / Q000378:metabolism*; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D010445:Peptide Elongation Factors; D011068:Poly G; D012343:RNA, Transfer; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D010444:Peptide Elongation Factor Tu