Effect of nucleoside modifications on the structure and thermal stability of Escherichia coli valine tRNA

Transfer RNA transcribed in vitro lacks the base modifications found in native tRNA. To understand the effect of base modifications on the structure of tRNA, the downfield region of the 1H NMR spectrum of in vitro transcribed E coli tRNAVal in aqueous phosphate buffer in the presence of excess Mg2+ was investigated. The resonances of all imino protons involved in hydrogen bonds in the helical stem regions and in tertiary interactions were assigned using two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) and one-dimensional difference nuclear Overhauser effect (NOE) methods. In addition, some aromatic C2 and C8 proton resonances as well as one amino proton resonance were assigned. The chemical shifts of the assigned resonances of unmodified E coli tRNAVal were compared with those of the native tRNA molecule under similar solution conditions. The similarity of the NMR data for unmodified and modified tRNA indicates that the in vitro transcribed tRNA has nearly the same solution structure as the native molecule in the presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interacting with) bases, indicating the possibility of local structural perturbations. The thermal stability of E coli modified and unmodified tRNAVal in the presence of Mg2+ was also investigated by analyzing the temperature dependence of the imino proton spectra. Several tertiary interactions involving modified nucleosides in native E coli tRNAVal are less stable in the absence of base modifications.