Abstract
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.
Highlights
Replicase withEF-Tu from a variety of sourcesI.n this Tu proteins [9,10,11]
Property results in an apparent increase in template kirromycin-sensitive wild type tufA gene product purified specificity; while wild tyQp/3e replicase will transcribe from strain LBE 2050
The results as themixture of mutant tufA and tu@ proteins purified from imply thatEF-Tu is involved in maintenance of enzyme the original double mutant strain LBE 2012 [9,10]
Summary
EF-Tu is unstable; it is rapidly inactivatedin the reac- geneous EF-Tu preparations: 1) kirromycin-resistant tufA tion mixture,even at temperatures as low as 20°C This gene product purifiedfrom strain LBE 2045 [10] and 2). Property results in an apparent increase in template kirromycin-sensitive wild type tufA gene product purified specificity; while wild tyQp/3e replicase will transcribe from strain LBE 2050. This strainwas prepared by transducpoly(C) and other synthetic RNA species, the mutant tion of a wild type tufA gene into strain LBE 2045.’. Protein or the mutant tu@ protein is apparently fully active, the enzyme made from kirromycin-resistant tufA protein is temperature sensitive.
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