Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s4U8–C13 photocrosslinking

摘要

Bacterial tRNAs frequently have 4-thiouridine (s⁴U) modification at position 8, which is adjacent to the C13-G22-m⁷G46 base triple in the elbow region of the tRNA tertiary structure. Irradiation with light in the UVA range induces an efficient photocrosslink between s⁴U8 and C13. The temperature dependence of the rate constants for photocrosslinking between the s⁴U8 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 s⁴U8 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 s⁴U8–C13 geometries. Analysis of tRNA^Val in buffers without Mg²⁺ indicate a smaller activation energy (∼13 kJ mol⁻¹) and a larger apparent s⁴U8–C13 distance (∼12 Å) compared to values for the same parameters in buffers with Mg²⁺ (∼26 kJ mol⁻¹ and 0.36 Å, respectively). These measurements are a quantitative indication of the strong constraint that Mg²⁺ imposes on the tRNA flexibility and structure.