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

Abstract

5-Fluorouracil is readily incorporated into active tRNA(Val) transcribed in vitro from a recombinant phagemid containing a synthetic E. coli tRNA(Val) gene. This tRNA has the expected sequence and a secondary and tertiary structure resembling that of native 5-fluorouracil-substituted tRNA(Val), as judged by 19F NMR spectroscopy. To assign resonances in the 19F spectrum, mutant phagemids were constructed having base changes in the tRNA gene. Replacement of fluorouracil in the T-stem with cytosine, converting a FU-G to a C-G base pair, results in the loss of one downfield peak in the 19F NMR spectrum of the mutant tRNA(Val). The spectra of other mutant tRNAs having guanine for adenine substitutions that convert FU-A to FU-G base pairs all have one resonance shifted 4.5 to 5 ppm downfield. These results allow assignment of several 19F resonances and demonstrate that the chemical shift of the 19F signal from base-paired 5-fluorouracil differs considerably between Watson-Crick and wobble geometry.

19F-Site-Specific-Labeled Nucleotides for Nucleic Acid Structural Analysis by NMR

“…A-U 5F to GÁU 5F wobble transitions observed in tRNA (Chu & Horowitz, 1989), a newly introduced U 5F 45 is deshielded and shifted by approximately 4.5 ppm to lower fields compared with U 5F 42 and U 5F 38 that adopt Watson-Crick geometries. While the C4]O of uridine accepts a hydrogen bond from the N6-H amino group of adenosine in Watson-Crick A-U base-pairing interactions, it is the uridine's C2]O that acts as a hydrogen bond acceptor within GÁU wobble base pairs.…”

Evidence for class-specific discrimination of a semiconserved base pair by tRNA synthetases

“…AlaRS concentrations of 5 and SO nM were used for the wild-type transcript and the N72 variants, respectively. Aminoacylation assays with purified E. coli valyl-tRNA synthetase (ValRS), which was a gift of Dr. Jack Horowitz, were done essentially as described (Chu & Horowitz, 1989). Assays with E. coli histidyl-tRNA synthetase (HisRS) were conducted as described (Francklyn & Schimmel, 1990) using 1 0 p M microhelixH" and 50-250 nM purified HisRS, which was a gift of Dr. Christopher Francklyn.…”

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).…”