Thermodynamic and kinetic framework of selenocysteyl-tRNASec recognition by elongation factor SelB

Abstract

SelB is a specialized translation elongation factor that delivers selenocysteyl-tRNA(Sec) (Sec-tRNA(Sec)) to the ribosome. Here we show that Sec-tRNA(Sec) binds to SelB.GTP with an extraordinary high affinity (K(d) = 0.2 pm). The tight binding is driven enthalpically and involves the net formation of four ion pairs, three of which may involve the Sec residue. The dissociation of tRNA from the ternary complex SelB.GTP.Sec-tRNA(Sec) is very slow (0.3 h(-1)), and GTP hydrolysis accelerates the release of Sec-tRNA(Sec) by more than a million-fold (to 240 s(-1)). The affinities of Sec-tRNA(Sec) to SelB in the GDP or apoforms, or Ser-tRNA(Sec) and tRNA(Sec) to SelB in any form, are similar (K(d) = 0.5 microm). Thermodynamic coupling in binding of Sec-tRNA(Sec) and GTP to SelB ensures at the same time the specificity of Sec- versus Ser-tRNA(Sec) selection and rapid release of Sec-tRNA(Sec) from SelB after GTP cleavage on the ribosome. SelB provides an example for the evolution of a highly specialized protein-RNA complex toward recognition of unique set of identity elements. The mode of tRNA recognition by SelB is reminiscent of another specialized factor, eIF2, rather than of EF-Tu, the common delivery factor for all other aminoacyl-tRNAs, in line with a common evolutionary ancestry of SelB and eIF2.

FIGURE 1.

Binding of SelB in the GTP-, GDP-, and apo-conformations to different forms of tRNA^Sec. A, binding of native Sec-tRNA^Sec (0.1 μm) to SelB·GTP (●), SelB·GDP (○), SelB-apo (□), and Sec-tRNA^Sec prepared by T7-RNA polymerase transcription to SelB-apo (■) measured by nitrocellulose filtration. As controls, Phe-tRNA^Phe interaction with SelB·GTP (♦) and Sec-tRNA^Sec binding to a filter-immobilized SelB·GTP without incubation (◇) are shown. B, binding of Ser-tRNA^Sec(Prf20) to SelB·GTP (●), SelB·GDP (■), apo-SelB (♦), and of tRNA^Sec(Prf20) to SelB·GTP (○), SelB·GDP (□), and apo-SelB (◇) measured by equilibrium fluorescence titrations. The fluorescence changes (16–33%) were normalized by putting the maximum signal change to 1.

FIGURE 2.

Kinetics of tRNA^Sec-SelB interactions. A, time courses of Sec-tRNA^Sec binding to SelB·GTP at 25 °C (●), 30 °C (○), 37 °C (■), and to SelB·GDPNP at 37 °C (□). B, concentration dependence of the apparent rate constant of Sec-tRNA^Sec binding to SelB·GTP at 25 °C. C, time courses of Sec-tRNA^Sec dissociation from SelB·GTP·Sec-tRNA^Sec at 25 °C (●), 30 °C (○), 37 °C (■), and from SelB·GDPNP·Sec-tRNA^Sec at 37 °C (□); k_off values are summarized in Table 2. D, time courses of dissociation of Sec-tRNA^Sec(Prf20) from the complex with SelB·GDP (1), Ser-tRNA^Sec(Prf20) from the complex with SelB·GTP (2), tRNA^Sec(Prf20) from SelB·GTP (3), SelB·GDP (4), and SelB-apo (5) at 25 °C. Traces in groups 1, 4, 5 and 2, 3 are indistinguishable.

FIGURE 3.

Ionic strength dependence of tRNA^Sec, Ser-tRNA^Sec, and Sec-tRNA^Sec binding to SelB·GTP. A, binding of SelB·GTP to Sec-tRNA^Sec in buffer B without NH₄Cl and with 150, 250, 350, 450, and 525 mm KCl (from top to bottom). B, dependence of K_d values on monovalent ion concentration of SelB·GTP·Sec-tRNA^Sec (●), SelB·GTP·Ser-tRNA^Sec(Prf20) (♦), and SelB·GTP·tRNA^Sec(Prf20) (◇). [M⁺] is the total concentration of monovalent cations in the reaction (in moles).

FIGURE 4.

Temperature dependence of tRNA^Sec, Ser-tRNA^Sec, and Sec-tRNA^Sec binding to SelB·GTP. A, binding of SelB·GTP to Sec-tRNA^Sec in buffer B with 430 mm KCl at 20, 25, 30, and 37 °C (from top to bottom). B, temperature dependence of the K_d values of SelB·GTP·tRNA^Sec(Prf20) (▴), SelB·GTP·Ser-tRNA^Sec(Prf20) (▵), and SelB·GTP·Sec-tRNA^Sec calculated from the rate constants of Sec-tRNA^Sec binding to and dissociation from SelB·GTP (Fig. 2) (●), or extrapolated (○) from the temperature dependence of the K_d values measured in buffer B with 480 mm KCl (◇), 430 mm KCl (♦), 380 mm KCl (□), and 330 mm KCl (■).

FIGURE 5.

A thermodynamic framework of the interactions between Sec-tRNA^Sec, SelB, and guanine nucleotides. K_d values of the respective interactions are indicated. The values for Sec-tRNA^Sec binding are from this report; for GTP/GDP binding to SelB are from Ref. ; and for GTP/GDP binding to SelB·Sec-tRNA^Sec are calculated from the law of mass action.