Qbeta-phage resistance by deletion of the coiled-coil motif in elongation factor Ts.

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.

Isolation of Qbeta polymerase complexes containing mutant species of elongation factor Tu.

The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.