Abstract
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.
Highlights
Elongation factor Tu (EF-Tu)1 promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome during the elongation step of protein synthesis in prokaryotes
Q Production in E. coli Mutant GRd.tsf Can Be Restored by Complementation with Wild-type elongation factor Ts (EF-Ts)—UY211 wild-type and GRd.tsf mutant cells were converted into FЈ strains, which was
Q-Phage Resistance—The present study demonstrates that the E. coli mutant GRd.tsf is completely resistant to Q-phage due to the inability of mutant EF-Ts without the coiled-coil motif to participate in the formation of a Q-polymerase complex
Summary
E. coli Strains—E. coli strain UY211 (ara, ⌬(lac-pro), nalA, thi) [30] and mutant strain GRd.tsf (ara, ⌬(lac-pro), nalA, thi, d.tsf) lacking the coiled-coil motif of EF-Ts (d.tsf) [29] were converted into Fϩ strains by conjugation with strain JC7782 (tolA, met, hsdS, lacY) FЈ(proABϩ, lacIq, lacZ⌬M15, Tn10(tetr)). Indicator strain GM1 was infected by adding 100 l of overnight culture prepared in glucose-M9 medium and 10 l of diluted phage-containing supernatant (or 250 l of undiluted supernatant from cultured GRd.tsf cells) to 5 ml of 0.75% (w/v) agar in LB medium at 42 °C. After co-expression of the native replicase subunit and the His-tagged EF-Tu in strains UY211 and GRd.tsf, cells were harvested by centrifugation (4,300 ϫ g, 20 min), resuspended in Q-replicase buffer (50 mM Tris-HCl, pH 7.8, 60 mM NH4Cl, 7 mM MgCl2 and 10% glycerol), and lysed by sonication. For purification of the Q-polymerase complex containing a Histagged Q-replicase subunit, cells of UY211 and GRd.tsf harboring plasmid pTrc99A-HisQ were grown at 37 °C in LB medium containing 200 g/ml ampicillin. The effect of guanine nucleotides on the stability of the polymerase core complexes was investigated by including either 0.1 mM GDP or 1 mM GTP with 60 M phospho-enol-pyruvate and 0.16 g/ml pyruvate kinase in the TBST buffer used for washing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
