Q beta replicase containing wild type and mutant tufA and tufB gene.
The protein synthesis elongation factor EF-Tu, complexed with EF-Ts, forms part of Q beta RNA replicase. In an effort to determine its function in the RNA synthesis reaction, we have developed procedures which allow us to replace the endogenous EF-Tu in purified Q beta replicase with EF-Tu from a variety of sources. In this communication we report purification of EF-Tu from strains containing (a) a wild type tufA gene only, (b) a kirromycin-resistant mutant tufA gene only, and (c) a kirromycin-resistant mutant tufA gene and a mutant tufB gene which codes for EF-Tu that does not bind ribosomes. When each of these EF-Tu preparations is inserted in Q beta replicase, the wild type tufA gene product and and the tufB gene product function appearently normally, but the kirromycin-resistant tufA gene product causes the formation of an altered enzyme. The Q beta replicase containing kirromycin-resistant EF-Tu is unstable; it is rapidly inactivated in the reaction mixture, even at temperatures as low as 20 degrees C. This property results in an apparent increase in template specificity; while wild type Q beta replicase will transcribe poly(C) and other synthetic RNA species, the mutant enzyme will do so only in the presence of Mn2+, which reduces template specificity. The kirromycin-resistant Q beta replicase will also transcribe Q beta RNA. The results imply that EF-Tu is involved in maintenance of enzyme structure, which, in turn, is implicated in template specificity.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005796:Genes; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D009154:Mutation; D010445:Peptide Elongation Factors / Q000096:biosynthesis*; D011728:Pyridones / Q000494:pharmacology; D011777:Q beta Replicase / Q000096:biosynthesis* / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000096:biosynthesis*; D013045:Species Specificity
D000900:Anti-Bacterial Agents; D010445:Peptide Elongation Factors; D011728:Pyridones; D008345:Manganese; D011777:Q beta Replicase; D012316:RNA Nucleotidyltransferases; D008274:Magnesium; C008536:mocimycin