Probing structural differences between native andin vitrotranscribedEscherichia colivaline transfer RNA: evidence for stable base modification-dependent conformers

Abstract

Structural differences between native (modified) and in transcribed (unmodified) Escherichia coli tRNA ^Val were explored by comparing their temperature- absorbance profiles as a function of magnesium ion concentration and by probing their solution conformation with single- and double-strand-specific endonucleases. In vitro transcribed tRNA ^Val has a less ordered structure as monitored by thermal melting profiles; its T _m is appreciably lower than that of native tRNA ^Val at all Mg ²⁺ concentrations. Structure probing experiments with nuclease S1 and ribonuclease V1 show that the unmodified tRNA ^Val transcript is more susceptible to nuclease attack at low Mg ²⁺ concentrations, particularly in the D- and T-loops, indicative of at least a partial disruption of D-loop/T-loop interactions. These experiments also provide evidence for temperature-dependent alternative conformations of the anticodon loop of native tRNA ^Val . Modified nucleosides are essential for the stability of these conformers; they cannot be detected in the unmodified in vitro transcript. The observations suggest that posttranscriptional modifications in tRNA allow the adoption of unique conformations and act to stabilize those that are biologically active.