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 = 6 X 10(-5) M. When
ppGpp is in twofold excess over
GTP and
EF-G is the rate-limiting component, the elongation rate is reduced twofold by
ppGpp.
EF-Tu is inhibited with
KI = 7 X 10(-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 = 4 X 10(-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 twofold 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.