Proton nuclear magnetic resonance of spin-labeled Escherichia coli tRNAf1MET.

Abstract

Thiouridine at position 8 (s4U8) of tRNAf-Met was spin-labeled with the nitroxide free radical, N-1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) bromacetamide, for proton nuclear magnetic resonance spectroscopic studies. The wellresolved methyl peak of ribothymidine is unperturbed, but the peak tentatively assigned to the C-5 methylene group of dihydrouridine is considerably broadened in spin-labeled tRNAf Met, Of the approximately 27 slowly exchanging protons observed in the region between 11 and 15 ppm downfield from 4,4-dimethyl-4-silapentane-1-sulfonic acid, the equivalent of about five protons apparently disappeared in spin-labeled tRNAf1Met. The well-resolved single proton at 14.8 ppm was missing not only in the paramagnetic species, but also in the diamagnetic reduced form of spin-labeled tRNAfMet, and was unequivocally identified as a hydrogen bond involving s4U8 by comparison of several forms of tRNAf Met specifically modified at s4U. Evidence that the perturbation of a second single proton resonance at 14.6 ppm (shift and broadening) is coupled to the loss of a tertiary hydrogen bond involving residue 8, arises from the same modified forms. The resolved resonances in the methyl and N-H regions, particularly the resonance at 14.6 ppm as well as the four N-bonded proton resonances at higher field which are broadened solely due to their proximity to the unpaired electron of the spin label, provide specific indicators of the geometry of tRNAfIMt structure in solution. Their observability by nuclear magnetic resonance spectroscopy opens up the possibility of monitoring distance changes among the base residues of spin-labeled tRNAf Met upon its interaction with aminoacyl-tRNA synthetase an other enzymes.The mechanism of recognition of specific tRNAs by their cognate aminoacyl-tRNA synthetases has been the subject of many investigations. For example, the relation of specificity to primary structure has been investigated by (i) comparison of sequences for homology in a group of tRNAs that are active substrates for a given synthetase (1, 2) and (ii) modifications of primary structure to distinguish essential from nonessential residues (3). Although the results have been suggestive, no universal principles of recognition have emerged. The conclusion that a specific tertiary structure of the molecule is necessary, if not sufficient, for recognition was early suggested by Fresco et al. (4) and emphasized by Cramer (1) and is now inescapable from the overwhelming amount of accumulated data.The three-dimensional structure derived from x-ray crystallographic analysis of yeast tRNAPhe (5,6) may prove to have specific as well as universal (7) The electron spin resonance (ESR) spectra of spin labels have been used to monitor the melting transition in aminoacylated tRNA labeled at the a-amino group of the aminoacyl moiety (12) and in yeast tRNAPhe labeled in the penultimate, residue of the 3' terminus in which C-C-A has been replaced by C-s2C-A (13). In the current experiments, NMR spectra have been recorded of Esc...

Conformational changes of transfer RNA. The role of magnesium(II)

“…Recently reported investigations raise legitimate questions in this regard. In particular, some of the resonance assignments used in our earlier study (Crothers et al, 1974) have become questionable because of the discovery of resonances associated with tertiary hydrogen bonds (Hilbers and Shulman, 1974; Wong and Kearns, 1974a;Daniel and Cohn, 1975). Of special interest for tRNArMeti is the careful study by Daniel and Cohn (1975), who showed that removal of the contaminating photocross-linkcd tRNAiMcti produced an NMR spectrum with much-improved peak resolution.…”

RNA structure

“…The lowest field region, n p.p.m. or more from DSS, contains 26-27 proton resonances in class I tRNAs corresponding to the number anticipated from a clover-leaf folding as well as about six additional tertiary resonances Daniel & Cohn, 1975). The exceptional resolution that can be attained under appropriate conditions is shown in Fig.…”

“…These are clearly revealed in the difference spectrum (C). The four resonances can be assigned rigorously to hydrogen-bonded ring protons of base pairs to the spin label (from Daniel & Cohn, 1975).…”

Biological Magnetic Resonance

“…The original assignment of the S4Vs . A14 base pair resonance has been confirmed by subsequent investigations (Daniel and Cohn, 1975;Reid and Robillard, 1975;Reid et al, 1975). Once it was demonstrated that at least one tertiary interaction could be observed by PMR, new experiments were designed to determine if there are other tertiary interactions which have low-field resonances.…”