Regulation of Turnover GTPase Activity of Elongation Factor G: the 30‐S‐Coupled and 30‐S‐Uncoupled Reactions

Abstract

The elongation factor G (EF‐G) GTPase activity exists in two differently regulated forms: one, dependent on both ribosomal subunits, is primarily expressed at high monovalent cation concentration; the other, dependent on the presence of the 50‐S subunit alone, becomes predominant at low concentrations of monovalent cations (30‐S‐uncoupled EF‐G GTPase). Both reactions show comparable Km values and responses to changes in [Mg2+] and pH, the 30‐S subunit increasing the affinity of EF‐G for the ribosome at both high and low salt concentration. At saturating [EF‐G], this causes an inhibition by the 30‐S subunit at low salt, whereas at higher ionic strengths the 30‐S subunit favors the turnover GTPase activity, apparently by counteracting the weakening effect of monovalent cations. By contrast, the EF‐G GTPase activity uncoupled from the 30‐S subunit steadily decreases with rising salt concentration. The action of the 30‐S subunit follows the predictions of the polyelectrolyte theory [Douzou, P. and Maurel, P. (1977) Proc. Natl Acad. Sci. U.S.A. 74, 1013]. As the pH is increased, the bell‐shaped K+ curve normally found with the 50‐S plus 30‐S subunits approaches the steadily descending curve observed with the 50‐S subunit alone, implicating an accumulation of negative charges in the vicinity of the binding site for the factor and/or the catalytic center. Thus these results suggest that the 30‐S subunit may expose a critical region of the ribosomal RNA, leading to a greater affinity for EF‐G. The 30‐S subunit exerts its effect at temperatures ranging from 20–65°C, and between pH 5 and 10.2. The pH optimum for association of the subunits and GTPase activity was the same (pH 8). Low salt concentration encourages formation of 70‐S ribosomes, while 50‐S and 30‐S subunits alone form multimers. NH4+ was the most efficient monovalent cation in 30‐S‐coupled EF‐G GTPase activity, followed by K+, Cs+, Li+ and Na+. The order of stimulation by divalent cations was Ba2+ > Sr2+ > Mg2−≥ Ca2+ > Mn2+. Treatment with the polyamine, spermidine, renders the GTPase activity dependent on both ribosomal subunits, insensitive to changes in monovalent cation concentration while suppressing the 30‐S‐uncoupled activity. This may represent a vital function in suboptimal growth conditions, when cells are unable to maintain an even intracellular K+ concentration.

Properties and regulation of the GTPase activities of elongation factors Tu and G, and of initiation factor 2

“…We noted early (96) that an increase in the EF-G to ribosome ratio augmented the 50S-dependent GTPase activity at low salt much more than that in the presence of both subunits at 80 mM NH +, and suspected therefore that the 30S subunit might act by increasing the affinity of the 50S subunit for EF-G 9 GTP. Figure 8 shows that this is indeed the case (83,84), not only at 2 mM NH4 + but also at 80 mM NH +. At 2 mM NH +, the midpoint of saturation with EF-G was a 22-fold excess over 50S, and a 2.4-fold excess over 50S plus 30S subunits.…”

“…At 80 mM NH4 +, the respective values for 50S and 50S plus 30S were a 60-and a tenfold excess of EF-G. Double reciprocal plots (83,84) showed that maximal velocities with saturating [EF-G] (--60 pmol GTP cleaved per pmo150S subunits in 1 min) were comparable for the tested assay systems except for the EF-G GTPase reaction dependent on boih ribosomal subunits at low salt; this was considerably slower. The Figure 8 (O) shows in fact that in this case high concentrations of EF-G failed to increase substantially the GTPase activity beyond a ratio of EF-G to ribosomes of approx.…”

“…This activity is progressively lost upon increasing the salt concentration. It does require the presence of divalent cations, having indeed the same Mg 2+ optimum as the reaction dependent on both subunits (84). Earlier claims of an EF-G GTPase promoted by 50S subunits alone were premature because these experiments were carried out either around 160 mM NH + (82, 95), a condition virtually abolishing this activity (83,84,(96)(97)(98), or at 60 mM NH4 + (94) where it should be largely inhibited.…”

“…It does require the presence of divalent cations, having indeed the same Mg 2+ optimum as the reaction dependent on both subunits (84). Earlier claims of an EF-G GTPase promoted by 50S subunits alone were premature because these experiments were carried out either around 160 mM NH + (82, 95), a condition virtually abolishing this activity (83,84,(96)(97)(98), or at 60 mM NH4 + (94) where it should be largely inhibited. Yet values of.36% (82), 39% (95) and 78% (94) of the activity with 70S ribosomes have been reported for the 50S-dependent EF-G GTPase activity, suggesting that the preparations of 50S subunits used in this work were contaminated with 30S subunits, which when dimerized sediment to the same position as 50S subunits (84 and are thus not detectable by centrifugation in sucrose gradients.…”

“…Increasing salt concentrations apparently counteract stabilization of the complex by 30S subunits, thereby raising the turnover of the GTPase activity up to an optimum beyond which instability of the complex itself becomes so prominent as to interfere with activity (84,99). As discussed in the section on ribosomal components, the 30S subunit may act by exposing or contributing negative charges (most probably phosphate groups of the ribosomal RNA), leading to efficient interaction with the factor.…”