Summary
The inhibition of elongation factors G, Tu and Ts by ppGpp was studied in vitro in a translation system with missense frequency and elongation rate similar to those in vivo. ppGpp inhibits EF-G with KI=6x10-5 M. When ppGpp is in twofold excess over GTP and EF-G is the rate-limiting component, the elongation rate is reduced two-fold by ppGpp. EF-Tu is inhibited with KI=7x10-7 M in the absence of EF-Ts. When EF-Ts is added, the binding of ppGpp to EF-Tu becomes successively weaker. 1/KI depends linearly on 1/[Ts] and the intercept at the abscissa gives KI=4x10-5 M. This reflects the binding of ppGpp to the binary TuTs complex. The slope reveals that the binding of EF-Ts to the TuMS binary complex is strong (10-6 M). ppGpp may thus inhibit the cycling of EF-Tu indirectly by the removal of the free EF-Ts by its adsorption to TuMS, as well as directly by simple binding to Tu. EF-Tu inhibition by ppGpp can be fully reversed by high levels of aminoacyl-tRNA only in the presence of EF-Ts and at low ribosomal activity. Our in vitro observations have been extrapolated to in vivo conditions with conclusions as follows: Under strong amino acid starvation ppGpp in two-fold excess over GTP cannot reduce significantly the elongation rate of ribosomes and thereby restore the errors to their normal levels as in the stringent response. Under weak starvation, in contrast, a significant rate reduction can be achieved by the trapping of EF-Ts in complex with TuppGpp.
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Rojas, AM., Ehrenberg, M., Andersson, S.G.E. et al. ppGpp inhibition of elongation factors Tu, G and Ts during polypeptide synthesis. Mol Gen Genet 197, 36–45 (1984). https://doi.org/10.1007/BF00327920
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DOI: https://doi.org/10.1007/BF00327920