Imino‐proton resonances of yeast tRNA^Phe studied by two‐dimensional nuclear Overhauser enhancement spectroscopy

Abstract

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H 2 0 solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G' . C7' and C 2 . G'' should be reversed).The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.Nuclear magnetic resonance spectroscopy has emerged as one of the most important tools for the structure elucidation of biomacromolecules in solution and for the study of their conformations and conformational dynamics [l]. For a time the full exploitation of the potential of the technique was severely impaired owing to difficulties in assigning individual resonances in the spectra to nuclei located at different sites in the molecules. In the last five years this situation has changed dramatically as a result of the application of the nuclear Overhauser effect (NOE) to biomacromolecules [2] and the development and application of two-dimensional Fourier transform techniques [3 -71.In NMR studies of tRNA the imino proton resonances have played a major role. These resonances are relatively well resolved and contain information about the helical structure and dynamics of base pairs in the molecule. Using one-dimensional (1 D) NOE techniques a complete spectral assignment of nearly all imino protons of tRNAPh" from yeast has been achieved [8 -111, while the imino-proton spectra of some other tRNAs have partly been characterized [12, 131. Notwithstanding these accomplishments two-dimensional NOE spectroscopy is, in principle, a more powerful and more efficient method for the detection of NOES between neighbouring protons in biological macromolecules [5 -7, 14-161 since the method can elucidate complete networks of spin connectivities in a single experiment. At this point, however, it should be realized that the vast majority of 2D NOE experiments reported up till now involve relatively small biomolecules (relative molecular mass less than lOOOO), of which the non-exchangeable protons (observable in D 2 0 ) are most easily examined. If water-exchangeable protons, e.g. the imino protons in nucleic acids, are also to be studied, an additional problem has to be overcome owing to the technical necessity of suppressing the huge resonance of water protons Previously we have reported that 2D NOE spectroscopy is indeed feasible for a molecule of the size of tRNA even when the aforementioned problems of large dynamic range are involved [16, 171. In order to take full advantage of th...

Time-resolved NMR monitoring of tRNA maturation

“…We next performed the chemical shift assignments of the imino groups of the two modified tRNA Phe samples, produced in E. coli and yeast, with the same approach used for the unmodified tRNA Phe sample. Imino protons assignments of the fully modified tRNA Phe are consistent with the ones reported by early NMR studies of this tRNA 36,37 . Chemical shifts assignments of the imino groups of the three samples are reported on 1 H– 15 N BEST-TROSY experiments measured in identical conditions in Fig.…”

Time-resolved NMR monitoring of tRNA maturation

“…We next performed the chemical shift assignments of the imino groups of the two modified tRNA Phe samples, produced in E. coli and yeast, with the same approach used for the unmodified tRNA Phe sample. Imino protons assignments of the fully modified tRNA Phe are consistent with the ones reported by early NMR studies of this tRNA (36,37). Chemical shifts assignments of the imino groups of the three samples are reported on 1 (Fig.…”

Influence of the polyamines spermine and spermidine on yeast tRNAphe as revealed from its imino proton NMR spectrum

“…Recently we analysed the effects of several naturally occurring polyamines on the thermal unfolding of phenylalanine and initiator tRNA from yeast by differential UV absorption [19]. In the present paper we extend our studies on polyamine-tRNA interaction by studying the imino proton part of the 500 MHz H NMR spectrum of phenylalanine tRNA from yeast for which reliable assignments of nearly all resonances have been obtained [20][21][22][23][24]. The most remarkable result from the present experiments is that polyamines are capable of catalysing the solvent exchange of several imino protons and at the same time stabilize the helix structure.…”