EFFECT OF NUCLEOSIDE MODIFICATIONS ON THE STRUCTURE AND THERMAL-STABILITY OF ESCHERICHIA-COLI VALINE TRANSFER-RNA

Transfer RNA transcribed in vitro lacks the base modifications found i n native tRNA. To understand the effect of base modifications on the s tructure of tRNA, the downfield region of the H-1 NMR spectrum of in v itro transcribed E coil tRNA(Val) in aqueous phosphate buffer in the p resence of excess Mg2+ Was investigated. The resonances of all imino p rotons involved in hydrogen bonds in the helical stem regions and in t ertiary interactions were assigned using two-dimensional nuclear Overh auser 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 a ssigned. The chemical shifts of the assigned resonances of unmodified E coli tRNA(Val) 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 t RNA has nearly the same solution structure as the native molecule in t he presence of excess Mg2+. The only significant differences were the chemical shifts of resonances corresponding to protons in (or interact ing with) bases, indicating the possibility of local structural pertur bations. The thermal stability of E coil modified and unmodified tRNA( Val) in the presence of Mg2+ was also investigated by analyzing the te mperature dependence of the imino proton spectra. Several tertiary int eractions involving modified nucleosides in native E coil tRNA(Val) ar e less stable in the absence of base modifications.