Perturbation of the tRNA tertiary core differentially affects specific steps of the elongation cycle

Abstract

The tRNA tertiary core region is important for both tRNA stability and activity in the translation elongation cycle. Here we report the effects of mutating each of two highly conserved base pairs in the tertiary core of Phe-tRNA(Phe), 18-55 and 19-56, on rate and equilibrium constants for specific steps of this cycle, beginning with formation of aminoacyl-tRNA.EF-Tu.GTP ternary complexs and culminating with translocation of A-site-bound peptidyl-tRNA into the P-site. We find that codon-dependent binding of aminoacyl-tRNA to the A/T-site and proofreading of near-cognate tRNA are sensitive to perturbation of either base pair; formation of the ternary complex and accommodation from the A/T to the A-site are sensitive to 18-55 perturbation only, and translocation of peptidyl-tRNA from the A- to P-site is insensitive to perturbation of either. These results underline the importance of the core region in promoting the efficiency and accuracy of translation, and they likely reflect different requirements for structural integrity of the core during specific steps of the elongation cycle.

FIGURE 1.

tRNA structure and the elongation cycle. A, crystal structure of yeast tRNA^Phe (Protein Data Bank code 1EHZ). Left, overall structure of the tRNA backbone (blue, 5′ half; orange, 3′ half) with nucleotide structures shown in the elbow region only. Right, blow-up of the elbow region, showing from left to right the base stacking of G19:C56, G57, G18:Ψ55, T54:m¹A58, and G53:C61. The D-loop and T-loop are shown in blue and orange, respectively. B, TC formation and the first elongation cycle of protein synthesis. Steps affected by mutation of the 18-55 and/or 19-56 bp(s) (see text) are indicated.

FIGURE 2.

Mutation effects on EF-Tu·GTP hydrolysis with cognate UUU codon. A, time course of GTP hydrolysis. Initiation complex, 0.4 μm. Ternary complex was formed by preincubating EF-Tu, Phe-tRNA^Phe, and GTP in the molar ratio 5:5:1. TC concentration was calculated using K_d values in Table 2. Curves are fit to single exponential equations. B, concentration dependence of the rate of GTP hydrolysis. The curves are fit to the Michaelis-Menten equation, yielding the following results: WT transcript, k_cat = 17 ± 2s^-1, K_m = 0.8 ± 0.2 μm; G18U/U55A, k_cat = 1.4 ± 0.1 s^-1, K_m = 0.6 ± 0.1 μm; C56A, k_cat = 6 ± 2s^-1, K_m = 2.4 ± 0.9 μm.

FIGURE 3.

Mutation effects on accommodation rate with cognate UUU codon. Accommodation was monitored by fMet-tRNA^fMet(prf) fluorescence change. Initiation complex, 0.4 μm; EF-Tu, 1 μm; Phe-tRNA^Phe, 0.2 μm; GTP, 200 μm. With one exception (U55A) curves are fit to a double exponential equation. For WT Phe-tRNA^Phe and most variants, the large decrease in the first reaction phase corresponds to accommodation, and it is followed by a small, slow decrease in the second phase of unclear origin, possibly because of a minor amount of misfolded tRNA. For U55A, which binds ternary complex slowly and with low stoichiometry, the first phase is not clearly distinguishable from the second, and results are fit with a single exponential. Relative amplitudes are observed changes for 0.2 μm ternary complex added and are thus corrected for incomplete TC formation, using the K_d values in Table 2. Monitoring of P-site tRNA^fMet does not permit observation of initial binding of Phe-tRNA^Phe to the A/T-site.

FIGURE 4.

Misreading assays with CUC codon. A, single turnover GTPase. B and C, kinetic profiles for dipeptide formation. Data from 0 to 30 s were taken using quenched flow. Initiation complex, 0.4 μm; EF-Tu, 0.4 μm; Phe-tRNA^Phe, 0.2 μm; GTP, 200 μm. Curves in A and C are fit to single exponential equations. Curves in B are fit to scheme 1 in D using Scientist, and setting k₁ equal to k′_GTPase determined in A (Table 3). Virtually identical fits were obtained if r values were set equal to R - 1 (Table 3). D, scheme 1, which accounts quantitatively for the results presented in B. k₃, determined by fitting, is equal to 0.005 s^-1 and reflects the amount of EF-Ts in the reaction mixture as a contaminant of EF-Tu (21). Adding EF-Ts (1 μm) to the reaction mixture (light blue trace in C) strongly accelerates dipeptide formation. k₂ is not evaluated but is ≫k₃. No reaction is observed when EF-Tu is omitted (C), showing that dipeptide formation does not proceed from nonenzymatic binding of Phe-tRNA.

FIGURE 5.

Apparent rate constants for dipeptide formation, UUU versus CUC. Relative apparent rate constants of dipeptide formation. White bars, UUU codon; black bars, CUC codon. Under the reaction conditions employed, the rate constants for WT Phe-tRNA^Phe are 2.2 s^-1 and 0.033 min^-1, respectively.

FIGURE 6.

Apparent rate constants for translocation with tRNA variants in the A- or P-sites. Comparison of the relative apparent rate constants for translocation of PRE-TR complexes containing either A-site (white bars) or P-site (black bars) variants. For A-site variants, translocation is measured by fMet-Phe-puromycin formation. Results for P-site variants are taken from Pan et al. (9), in which translocation was measured by the fluorescence change in fMet-Phe-tRNA^Phe(prf). Both assays of translocation give essentially the same results.