Abstract
Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).
Highlights
The equilibrium dissociation constant governing the affinity of E. coli Elongation factor (EF)-Tu for GDP is 8 nM, compared with 0.3 M for GTP (1)
Equilibrium Dissociation Constant for the Release of aatRNA from the Ternary Complex—The function of EF-Tu is to guide aa-tRNA into the A-site of the ribosome. This step requires the formation of a ternary complex composed of EF-Tu, aa-tRNA, and GTP
Since EF-Tumt forms functional ternary complexes with aa-tRNAs from E. coli, we used E. coli PhetRNAPhe to measure the equilibrium dissociation constant governing the ternary complex formed with EF-Tumt (13)
Summary
The equilibrium dissociation constant governing the affinity of E. coli EF-Tu for GDP is 8 nM, compared with 0.3 M for GTP (1). No elongation factor equivalent to EF-Ts has been identified in yeast mitochondria or in the complete sequence of the yeast genome, perhaps reflecting the observation that yeast EF-Tumt has a very low affinity for guanine nucleotides (10). Unlike the E. coli EF-Tu1⁄7Ts complex, the bovine liver EFTu1⁄7Tsmt complex cannot be dissociated in the presence of high concentrations of guanine nucleotides (11). This observation is due, at least in part, to a low affinity of EF-Tumt for GDP and GTP. Mitochondrial Elongation Factor Tu sociation constants governing the release of aa-tRNA from the ternary complex and the dissociation of the EF-Tu1⁄7Tsmt complex are reported here
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