Conformation of Charged and Uncharged tRNA ^Phe

Abstract

The effect of aminoacylation on the conformation of yeast tRNAPhe was investigated by highresolution (300 MHz) proton nuclear magnetic resonance (NMR) spectroscopy. Resonances in the low-field (-11 to -15 ppm) region of the spectra are due to ring NH protons of Watson-Crick base pairs, and to a very high degree of approximation (within 0.05 ppm) the low-field spectra of tRNAPhe and phenylalanyl-tRNAPhe are identical. From this observation and analysis of the low-field NMR spectra we conclude that the secondary structures of the two tRNAs are identical with respect to base-pairing schemes and interbase distances in the helical region (0.1-0.2 A). Several tertiary structural features, including conformation of the dihydro-U loop, conformation of the minor loop, relative orientations of the acceptor and the TV&C stems, dihydro-U and anticodon stems, and probably conformation of the anticodon loop are shown to be the same in tRNAPhe and phenylalanyl-tRNAPhe. Our results leave little remaining opportunity for changes in tertiary structure that would not have been observed by the NMR method.In order to account for the role that tRNA plays in protein synthesis, it has been suggested that aminoacylation (or deacylation) induces a change in the secondary and/or tertiary structure of the tRNA (1-3). During the past 7 years, there have been numerous attempts to investigate this problem, but in each case, the methods used were limited in the information they provided and the results were often conflicting. Sarin and Zamecnik (1), for example, reported significant differences between the optical rotatory dispersion of unfractionated aminoacyl-tRNA and tRNA, but Adler and Fasman (4) used purified tRNAs and found no difference in the circular dichroism of aminoacyl-tRNA and tRNA. Gantt, Englander, and Simpson (5) found differences between the hydrogen-exchange properties of unfractionated Escherichia coli tRNA before and after aminoacylation, but the differences in the number of exchangeable protons detected by the tritium exchange technique was small (order of 4%). In a subsequent study of the hydrogen-exchange properties of E. coli tRNAfMet, Englander, Kallenbach, and Englander (6) were unable to detect any difference between aminoacylated and deacylated tRNA.Hanggi and Zachau (7) were unable to detect any difference in gel filtration or in partial nuclease digestion of tRNA and aminoacylated tRNA, and concluded that there were no gross conformational changes in tRNA induced by aminoacylation. Cohn, Danchin, found that binding of Mn2+ to tRNA was increased by aminoacylation of tRNA, but did not speculate on possible conformational changes that might be involved. Danchin and Grunberg-Manago (9) found that the binding of oligo (C) to unfractionated E. coli tRNA, purified yeast tRNAPhO, and E. coli tRNAval is stronger when the tRNAs are aminoacylated, and concluded from this that a region of the tRNA containing at least two guanine residues was exposed in the aminoacylated tRNAs but not in the uncharged tRNA. They furth...

Spin-labelled nucleic acids

“…The data on this problem obtained by different techniques have been rather ambiguous. The integral methods characterizing tRNA as a whole did not reveal essential changes in the molecular structure during aminoacylation (Hanggi & Zachau, 1971;Wong, Reid & Kearns, 1973). In contrast, the techniques that enabled one to follow local sites of the structure detected such changes (Watanabe & Imahori, 1971;Thomas et al 1973).…”

Changes in the solution structure of yeast phenylalanine transfer ribonucleic acid associated with aminoacylation and magnesium binding

“…Subsequent experiments, using techniques more sensitive to tRNA substructure, have similarly produced conflicting results. Analysis of aminoacylated tRNA by low-field NMR, a technique sensitive to secondary and tertiary hydrogen bonding, has shown no differences for aminoacylated and nonacylated yeast tRNAPhe in a pH 5 solution condition (Wong et al, 1973); a second study performed at pH 6.6 by another laboratory showed changes occurring in the region around ribothymidine-54 in the loop (Davanloo et al, 1979).…”

Carbodiimide modification analyse of aminoacylated yeast phenylalanine tRNA: evidence for change in the apex region

“…However, the reagent used required that modification be performed at low pH (5.0-6.0) and very high ionic strength (>1.OM), conditions that may prevent the pertinent structural changes from taking place. In an NMR analysis, no difference was observed in the spectra of tRNAPhe and phe-tRNAPhe (51). However, that analysis was performed at pH 5, again a condition that may not allow an acylationdependent change to occur.…”