Inhibition of transfer RNA function by replacement of uridine and uridine-derived nucleosides with 5-fluorouridine

Revtovich

2016

mSphere

Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans. This method led to the unexpected discovery that addition of a modified nucleotide, 5-fluorouridine, disrupted bacterial RNA metabolism and inhibited synthesis of pyoverdine, a critical toxin. Our results demonstrate that this compound specifically functions as an antivirulent.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A High-Content, Phenotypic Screen Identifies Fluorouridine as an Inhibitor of Pyoverdine Biosynthesis and Pseudomonas aeruginosa Virulence

Revtovich

2016

mSphere

“…The 19F-labeled tRNAs represent ideal subjects for investigation by 19F NMR because they remain fully active in protein synthesis in vitro despite having essentially all their uracil and uracilderived modified bases replaced by 5-fluorouracil (20)(21)(22)(23). Fluorouracil is incorporated into all stems and loops of the tRNA, where each residue can serve as a site-specific reporter of structural changes in its vicinity.…”

Section: Introductionmentioning

confidence: 99%

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

1986

Self Cite

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.

“…However, several (FUra)tRNAs are aminoacylated at significantly lower rates and to lower extents than their normal counterparts (Ramberg et al, 1978). Lysine tRNA is the most inhibited, being only 6% as active as normal tRNA^ys, FUra-substituted Lys-tRNA^ys is also impaired in its ability to bind to ribosomes and function in polypeptide synthesis (Ramberg et al, 1978).…”

Section: Interaction With Proteinsmentioning

confidence: 99%

“…Aminoacylation with aspartate, glutamate, glutamine, and histidine is also inhibited. All these analogue-substituted tRNAs with reduced activity contain a 5-fluorouridine residue in the second position of their anticodons, and this substitution may be, at least in part, responsible for their lowered activities (Ramberg et al, • 1978).…”

Section: Interaction With Proteinsmentioning

confidence: 99%

19F NMR studies of 5-fluorouracil-substituted Escherichia coli transfer RNAs: solution structure and codon-anticodon interaction

Gollnick

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