19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

Horowitz, Jack; Ofengand, James; Daniel, Walter E.; Cohn, Mildred

doi:10.1016/s0021-9258(17)40283-3

Cited by 35 publications

(21 citation statements)

References 8 publications

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“…Neither of these tRNAs could be cross-linked. The slight activity of the FU-tRNAVal is consistent with the residual 10% unsubstituted uridine-derived residues remaining in the preparation (Horowitz et al, 1977). Figure 7 also verifies the strong dependence of covalent linking on the presence of a polynucleotide messenger RNA while Table III shows that any polynucleotide will not suffice for covalent linking, but only one containing the appropriate codons.…”

Section: Resultssupporting

confidence: 60%

Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

Ofengand¹,

Liou²,

Kohut³

et al. 1979

Biochemistry

109

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The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.

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Section: Resultssupporting

confidence: 60%

Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

Ofengand¹,

Liou²,

Kohut³

et al. 1979

Biochemistry

109

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“…Given its attractive spectroscopic properties, 19 F was incorporated into RNA for NMR studies in the 1970s (7)(8)(9). Since then, 19 F has been successfully incorporated into DNA and RNA oligonucleotides for NMR analysis and used to probe RNA and DNA structure, conformational exchange, and macromolecular interactions (10,11).…”

Section: Introductionmentioning

confidence: 99%

Solution NMR readily reveals distinct structural folds and interactions in doubly ¹³ C- and ¹⁹ F-labeled RNAs

et al. 2020

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RNAs form critical components of biological processes implicated in human diseases, making them attractive for small-molecule therapeutics. Expanding the sites accessible to nuclear magnetic resonance (NMR) spectroscopy will provide atomic-level insights into RNA interactions. Here, we present an efficient strategy to introduce 19F-13C spin pairs into RNA by using a 5-fluorouridine-5′-triphosphate and T7 RNA polymerase–based in vitro transcription. Incorporating the 19F-13C label in two model RNAs produces linewidths that are twice as sharp as the commonly used 1H-13C spin pair. Furthermore, the high sensitivity of the 19F nucleus allows for clear delineation of helical and nonhelical regions as well as GU wobble and Watson-Crick base pairs. Last, the 19F-13C label enables rapid identification of a small-molecule binding pocket within human hepatitis B virus encapsidation signal epsilon (hHBV ε) RNA. We anticipate that the methods described herein will expand the size limitations of RNA NMR and aid with RNA-drug discovery efforts.

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“…To help resolve these differences and to better understand the mechanism of EB binding, the interaction of EB with Escherichia coli tRNA Val and an RNA minihelix corresponding to the acceptor stem and T-arm of tRNA Val was examined by 1 H and 19 F NMR. 19 F NMR is a powerful probe of the solution structure of tRNA labeled by incorporation of 5-fluorouracil (FUra) (20)(21)(22)(23)(24)(25)(26)(27). The 14 FUra residues in FUra-substituted E.coli tRNA Val [(FUra)tRNA Val ] are distributed throughout all stems and loops of the molecule (Fig.…”

Section: Introductionmentioning

confidence: 99%

Localization of the Major Ethidium Bromide Binding Site on tRNA

Chu¹,

Liu²,

Horowitz³

1997

Nucleic Acids Research

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Binding of ethidium bromide to Escherichia coli tRNAVal and an RNA minihelix based on the acceptor stem and T-arm of tRNAVal was investigated by 19F and 1H NMR spectroscopy of RNAs labeled with fluorine by incorporation of 5-fluorouracil. Ethidium bromide selectively intercalates into the acceptor stem of the tRNAVal. More than one ethidium bromide binding site is found in the acceptor stem, the strongest between base pairs A6:U67 and U7:A66. 19F and 1H spectra of the 5-fluorouracil-substituted minihelix RNA indicate that the molecule exists in solution as a 12 base-paired stem and a single-stranded loop. Ethidium bromide no longer intercalates between base pairs corresponding to the tRNAVal acceptor stem in this molecule. Instead, it intercalates between base pairs at the bottom of the long stem-loop structure. These observations suggest that ethidium bromide has a preferred intercalation site close to the base of an RNA helical stem.

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19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

Cited by 35 publications

References 8 publications

Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

Solution NMR readily reveals distinct structural folds and interactions in doubly ¹³ C- and ¹⁹ F-labeled RNAs

Localization of the Major Ethidium Bromide Binding Site on tRNA

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19F nuclear magnetic resonance of 5-fluorouridine-substituted tRNA1Val from Escherichia coli.

Cited by 35 publications

References 8 publications

Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

Covalent crosslinking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid

Solution NMR readily reveals distinct structural folds and interactions in doubly 13 C- and 19 F-labeled RNAs

Localization of the Major Ethidium Bromide Binding Site on tRNA

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Solution NMR readily reveals distinct structural folds and interactions in doubly ¹³ C- and ¹⁹ F-labeled RNAs