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

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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.