Fluorine-19 nuclear magnetic resonance study of codonanticodon interaction in 5-fluorouracil-substitutedE. colitransfer RNAs

Abstract

Codon-anticodon interaction was investigated in fully active 5-fluorouracil-substituted E. coli tRNAyal (anticodon FAC) by 19F NMR spectroscopy.Binding of the codon GpUpA results in the upfield shift of a 19F resonance at 3.9'ppm in the central region of the 19F NMR spectrum, whereas trinucleotides not complementary to the anticodon have no effect. The same 19F resonance shifts upfield upon formation of an anticodon-anticodon dimer between the 19F-labeled tRNA and E. coli tRNA4Yr (anticodon QUA). These results permit assignment of the peak at 3.9 ppm to the 5-fluorouracil at position 34 in the anticodon of fluorouracil-substituted tRNAyal. The methionine codon ApUpG also causes a sequence-specific upfield shift of a peak in the central part of the 19F NMR spectrum of fluorinated E. coli tRNAMet.However, ApUpG has no effect on the 19F spectrum of I9F-labeled E. coli tRNApet, indicating possible conformational differences between the anticodon loop of initiator and chain-elongating methionine tRNAs. 19F NMR experiments detect no binding of C GpA A to the complementary F FpC G (replaces Tp'PCpG) in the T-loop of 9-fluorouracil-substituted tRNAyal, in the presence or absence of codon, suggesting that the tertiary interactions between the T-and D-loops are not disrupted by codon-anticodon interactions.

Conformational heterogeneity of the protein-free human spliceosomal U2-U6 snRNA complex

“…We then exploited different chemical shifts of 5-19 F-incorporated pyrimidine nucleotide, in either a single-or doublestranded region of RNA, which has previously been used to probe the secondary structure of RNA (Horowitz et al 1977;Marshall and Smith 1977;Gollnick et al 1986;Chu et al 1992;Kanyo et al 1996;Sahasrabudhe and Gmeiner 1997;Arnold and Fisher 2000;Hammann et al 2001;Olejniczak et al 2002;Hennig et al 2007;Puffer et al 2009) to demonstrate the presence of an alternative conformation of U2-U6 snRNA complex conclusively. The advantage of observing the 19 F nucleus by NMR is the broader range and the greater sensitivity of fluorine chemical shifts in response to the local environment as compared to those of hydrogen because the fluorine nucleus is surrounded by nine electrons vs. a single electron in hydrogen.…”

19F NMR of 5-fluorouracil-substituted transfer RNA transcribedin vitro: resonance assignment of fluorouracil-guanine base pairs

“…At high magnesium ion concentrations, in vitro transcribed (FUra)tRNAVaI and that synthesized in vivo have very similar secondary and tertiary structures, as shown by the similarity of their '9F NMR spectra (Figure 3a and b). Because the chemical shift of the fluorine nucleus is very sensitive to changes in tRNA conformation (4)(5)(6)(7)(8), any significant structural variations would be reflected in spectral changes. The small chemical shift changes of peaks E and K in the spectrum of the fluorinated in vitro transcript may be due to the absence of m7G and m6A in this tRNA; these are the only two modified bases present in native (FUra)tRNAVal prepared from E. coli cells (9).…”

“…These investigations have employed a number of NMR techniques involving protons and other magnetic nuclei, including 13C, 31p, and 15N (reviewed in 1). Another useful NMR label for the study of biological systems is fluorine-19, and we have employed 19F NMR as a structural probe of tRNAs labeled by incorporation of 5-fluorouracil (FUra) (2)(3)(4)(5)(6)(7)(8). The 19F nucleus has the advantages of 100% abundance, a large range of chemical shifts, and high sensitivity (83 % that of 'H).…”

“…Spectra of three purified 5-fluorouracil-substituted E. coli tRNAs, tRNAVal, tRNAfMet and tRNAmMet, have been reported (2,3,8), and these show resolved resonances for each of the incorporated FUra residues. Oligonucleotide binding, chemical modification, and nuclear Overhauser effect studies were used to assign several of the 14 peaks in the 19F NMR spectrum of tRNAvSl (4,5,7,8). To verify and complete the assignments, we have prepared FUra-substituted tRNAval by in vitro transcription of a synthetic E. coli tRNAval gene, using a method similar to that recently reported by Sampson and Uhlenbeck (12).…”

Investigations on Fluorine‐Labeled Ribonucleic Acids by19F NMR Spectroscopy

“…Pioneering studies by Horowitz and coworkers on fluorinated tRNAs were commenced about 30 years ago [23][24][25][26][27][28][29][30][31][32][33][34][35]. In these experiments, tRNAs were in-vitro transcribed in the presence of 5-fluorouridine triphosphates, such that all uridine positions were replaced by the fluorinated analogue (Figure 1.10).…”