Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s(4)U8-C13 photocrosslinking

Abstract

Bacterial tRNAs frequently have 4-thiouridine (s(4)U) modification at position 8, which is adjacent to the C13-G22-m(7)G46 base triple in the elbow region of the tRNA tertiary structure. Irradiation with light in the UVA range induces an efficient photocrosslink between s(4)U8 and C13. The temperature dependence of the rate constants for photocrosslinking between the s(4)U8 and C13 has been used to investigate the tRNA conformational energy and structure in Escherichia coli tRNA(Val), tRNA(Phe), and tRNA(fMet) under different conditions. Corrections have been made in the measured rate constants to compensate for differences in the excited state lifetimes due to tRNA identity, buffer conditions, and temperature. The resulting rate constants are related to the rate at which the s(4)U8 and C13 come into the alignment needed for photoreaction; this depends on an activation energy, attributable to the conformational potential energy that occurs during the photoreaction, and on the extent of the structural change. Different photocrosslinking rate constants and temperature dependencies occur in the three tRNAs, and these differences are due both to modest differences in the activation energies and in the apparent s(4)U8-C13 geometries. Analysis of tRNA(Val) in buffers without Mg(2+) indicate a smaller activation energy (~13 kJ mol(-1)) and a larger apparent s(4)U8-C13 distance (~12 A) compared to values for the same parameters in buffers with Mg(2+) (~26 kJ mol(-1) and 0.36 A, respectively). These measurements are a quantitative indication of the strong constraint that Mg(2+) imposes on the tRNA flexibility and structure.

FIGURE 1.

Location of the s⁴U8–C13 photocrosslinking site in the tRNA secondary and tertiary structures. (A) Cloverleaf structure and sequence of E. coli tRNA^Val with the s⁴U8 and C13 nucleotides indicated. (B) Location of U8 and C13 in the yeast tRNA^Phe tertiary structure. U8 is not thiolated in yeast tRNA^Phe.

FIGURE 2.

Analysis of kinetics of crosslink formation. (A) Gel electrophoresis separation of irradiated tRNA. tRNA^fMet and tRNA^Val were 3′ end exchange-labeled and irradiated in HiFi buffer with wavelengths >320 nm for the indicated times at 0°C and 45°C. Bands 1 and 2 separate after electrophoresis on 8% polyacrylamide urea gel and contain the uncrosslinked and crosslinked tRNA as confirmed by RNA sequencing (Supplemental Material). (B) Time course of photocrosslinking for tRNA^fMet and tRNA^Val. Plot of fraction crosslinked versus irradiation time (in seconds) for tRNA^fMet and tRNA^Val at 0°C and 45°C as indicated in the figure. The irradiations were done in a buffer containing 20 mM Tris (pH 7.5), 100 mM NH₄Cl, and 20 mM Mg²⁺. The curves were fitted using parameters for the first order rate constants and for the total amount reactive. The total extent of crosslinking for tRNA^fMet and tRNA^Val is 0.80 ± 0.01 and 0.72 ± 0.02, respectively. (C) First order plot of rate of photocrosslinking. The logarithm of the fraction of tRNA unreacted for the s⁴U8×C13 photocrosslink in tRNA^fMet and tRNA^Val is plotted versus time for the irradiations at 0°C and 45°C.

FIGURE 3.

Temperature dependence of rate constants for formation of the s⁴U×C13 crosslink in tRNA^fMet, tRNA^Val, and tRNA^Phe. (A) First order rate constants calculated from 1-min irradiations at different temperatures and corrected for changes in excited state lifetimes. The fitted lines are exponential functions. Uncertainty bars are standard deviations of measurements at each temperature. (B) Arrhenius plots for the corrected first order rate constants. The fitted lines are linear regression lines.

FIGURE 4.

Dependence of the corrected rate constants, energies of activation, and calculated parameters on NH₄OAc and Mg²⁺ concentrations for the tRNA^Val s⁴U8×C13 crosslink. (A) Dependence of the corrected first order rate constants on NH₄ ⁺ and Mg²⁺ concentrations. All data in this and subsequent panels are from Table 1. (B) Dependence of activation energies on NH₄ ⁺ and Mg²⁺ concentrations. (C) Dependence of the s⁴U8–C13 apparent distance on NH₄ ⁺ and Mg²⁺ concentrations. (D) Dependence of force constants on NH₄ ⁺ and Mg²⁺ concentrations. Note that there is a 100-fold change in scale in the left and right parts of panel D. For all experiments, the dependence on different NH₄ ⁺ concentrations was determined in the presence of 20 mM Tris (pH 7.5) and the dependence on different Mg²⁺ concentrations was determined in the presence of 20 mM Tris (pH 7.5), 100 mM NH₄Cl.