This paper describes the isolation of 3-fluo- MATERIALS AND METHODSThe E. colh strain used throughout these experiments is CSH46 (same as M96) (9). This strain requires proline and thiamine, is unable to grow on arabinose or lactose, and is lysogenic for a temperature-inducible prophage (XcI857, St68, hS0dlacIZ) which in turn carries part of the lac operon. The lac region contains a mutation in the Z gene (U118) and has the I (SQ) overproducing promoter.Media. Rich medium, when used, was LB (9). Minimal medium was M9 (9) supplemented with glucose (1%), thiamine(1 ug/ml) and the naturally occurring L amino acids (20.ug/ml, or 0.2 mM) except tyrosine, tryptophan, and phenylalanine. Tryptophan and phenylalanine were present at 1 mM.DL-3-Fluorotyrosine was synthesized starting with 3-fluoro-p-anisaldehyde (Aldrich Chemical Co.) as described in ref. 10. The 3-fluorotyrosine obtained was characterized by its-infrared and ultraviolet spectra, and by the 19F NMR spectrum at neutral and alkaline pH-. No tyrosine contamination could be found when the 3-fluorotyrosine was chromatographed on cellulose plates in isopropanol:NH40H (17%):H20, volume ratio 4:2:1, or electrophoresed at pH 9.2 in 0.05 M NH4HCO3 buffer on Whatman 3MM paper. Separation in the electrophoresis occurs because the pK of the 3-fluorotyrosine phenolic proton is 8 rather than 10.5 for tyrosine. 3-Fluoro[3H]tyrosine and [3H]tyrosine were made by exchanging the 3H from 3H20 onto the a position (11).Purification of the 3-fluorotyrosine lac repressor, isopropyl-fl-D-thiogalactoside (IPTG) binding assays, and operator DNA binding assays follow published procedures for normal lac repressor (9,(12)(13)(14). The 3-fluorotyrosine lac repressor had about 35% activity for operator binding, comparable to published results with normal lac repressor (12, 13). The absorption coefficient was determined by comparing a biuret protein measurement (15) of the 3-fluorotyrosine lac repressor with a standard curve obtained for normal lac repressor. Both proteins were judged to be greater than 98% pure from gel electrophoresis (16). Overall yields of 3-fluorotyrosine lac repressor from the phosphocellulose step (12) have been about 40-50% of the

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...

A study of the exchangeable ring nitrogen protons in aqueous solutions of oligonucleotide complexes involving Watson-Crick base pairs as well as Hoogsteen pairs and other nonclassical hydrogen bonding schemes shows that resolvable resonances in the low-field (-10 to -16 ppm from sodium 4,4-dimethyl-4-silapentanesulfonate) region can be detected in a variety of structures other than double stranded helices. Ring nitrogen proton resonances arising from the following hydrogen-bonding situations are reported: (1) AT and GC Watson-Crick base pairs in a self-complementary octanucleotide, dApApApGpCpTpTpT; (2) U-A-U base triples in complexes between oligo-U15 and AMP; (3) C-G-C+ base triples in complexes between oligo-C17 and GMP at acid pH; (4) s4U-A-s4U base triples in complexes between oligo-s4U15 and AMP, all of which involve both Watson-Crick and Hoogsteen base pairing to form triplexes; (5) C-C+ base pairing between protonated and unprotonated C residues in oligo-C17 at acid pH; and (6) I4 base quadruples in the four strand association among oligo-I at high salt. The behavior of the dA3G-CT3 helix is consistent with both fraying of the terminal base pairs and presence of intermediate states as the helix opens. In the monomer-oligomer complexes, under the conditions used here, the exchange appears to be governed by the dissociation rate of monomer from the complex. These findings suggest that those tertiary structure hydrogen bonds in tRNA involving ring nitrogen protons should have representative resonances in the low-field (11-16 ppm) proton NMR region in H2O.

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The properties of Escherichia coli tRNAMet f1 and tRNAMet f3 that differ by only one base change, m7G to A at position 47, have been compared structurally by proton magnetic resonance and functionally by the aminoacylation reaction. The NMR spectra of the two tRNA species in the region between 0 and 4 ppm below 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) (methyl and methylene region) were the same except for the absence of the lowest field peak at 3.8 ppm in tRNAMet f3, thus unequivocally identifying this resonance at the methyl group of m7G47 of tRNAMet f1. The same resonance disappears in tRNAMet f1 spin-labeled at s4U8 and reappears in the diamagnetic reduced spin-labeled tRNAMet f1 from which the average distance between the spin-label and the methyl protons of m7G is estimated to be less than 15 A. The proximity of m7G47 but not T55 to s4U8 in the structure of E. coli tRNAMet f1 in solution is consistant with the crystallographic model for yeast tRNAPhe. A spectral comparison of the hydrogen-bond regions (11-14 ppm below DSS) of tRNAMet f1 and tRNAMet f3 reveals major shifts of four resonances previously assigned to tertiary hydrogen bonds. Of the four, the one at lowest field (14.8 ppm) had been assigned by chemical modification to the tertiary (s4U8-A14) hydrogen bond and the one at 13.3 ppm had been tentatively assigned to the tertiary hydrogen bond G23-m7G47 of the 13-23-47 triple. A more positive assignment of the G23-m7G47 at 13.3 ppm could be made from the additional evidence that this resonance, which was first observed in the difference spectrum between spin-labeled tRNAMet f1 and its reduced form, is the only one missing in the analogous difference spectrum of tRNAMet f3. At low ionic strength and in the absence of magnesium ions, the differences in the hydrogen-bonded region of the NMR spectra of tRNAMet f1 and tRNAMet f3 are much greater than in the presence of magnesium ions. The optimal magnesium concentration required for maximal initial velocities is also higher for tRNAMet f3 than for tRNAMet f1. The perturbation caused by the spin-label in destabilizing hydrogen bonds in the region between 13 and 14 ppm is greater for tRNAMet f3 than tRNAMet f1 but the distance relations for the hydrogen bonds in the region between 12 and 13 ppm (the major paramagnetic perturbations) are conserved in the two species. The disruption of one hydrogen bond relative to native tRNAMet f1 either by spin-labeling (s4U8-A14) or by substitution of m7G by A in tRNAMet f3 has little effect on the aminoacyl acceptor activity or the velocity of the aminoacylation reaction at optimal magnesium concentration, but the absence of both tertiary hydrogen bonds in the augmented D-helix region in the spin-labeled tRNAMet f3 results in approximately 60% reduction both in acceptance activity and in initial velocity of the aminoacylation reaction.