Fluorescent labeling of tRNA dihydrouridine residues: Mechanism and distribution

Abstract

Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward reduction by NaBH 4 , their abundance within prokaryotic and eukaryotic tRNAs, and the biochemical functionality of the labeled tRNAs so produced. Interpretation of experiments employing labeled tRNAs can depend on knowing the distribution of dye among the DHU positions present in a labeled tRNA. Here we combine matrixassisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis of oligonucleotide fragments and thin layer chromatography to resolve and quantify sites of DHU labeling by the fluorophores Cy3, Cy5, and proflavin in Escherichia coli tRNA Phe and E. coli tRNA Arg . The MALDI-MS results led us to re-examine the precise chemistry of the reactions that result in fluorophore introduction into tRNA. We demonstrate that, in contrast to an earlier suggestion that has long been unchallenged in the literature, such introduction proceeds via a substitution reaction on tetrahydrouridine, the product of NaBH 4 reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA.

Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis

“…After washing away unbound complexes, collection of real-time translation traces began 10 s prior to injecting 10 nM fluorescent-labeled ternary complexes, 2 μM EF-G and 2 mM GTP into the flow cell. Fluorescent-labeled ternary complexes were preformed from EF-Tu, GTP, and charged tRNAs labeled with either Cy3 or Cy5 at dihydrouridine positions in the D loop (57,58). Recording continued Fig.…”

Tb3+-tRNA for LRET Studies of Protein Synthesis

“…5,6 Such labeling proceeds via a substitution at the THU residues to yield N -substituted tetrahydrocytidine (THC) residues within the tRNA. Previous work 6 showed that Cy3 hydrazide labeled Phe-tRNA Phe (reduced) at both positions 16 and 20 to near equal extents (relative labeling 1.0:1.2, respectively) and we assume a similar distribution is obtained for labeling of tRNA Phe (reduced) by VI . In the present case, the overall labeling stoichiometry was 0.4 V /tRNA Phe .…”

“…In this prior work, fl-tRNAs were synthesized by condensing fluorescent weak-base amines, including hydrazides, with the tetrahydrouridine (THU) residues in bulk tRNA that are formed by specific reduction of dihydrouridine (DHU) residues within the D-loops of tRNA, as described. 4–6 The FRET signal generated using bulk fl-tRNAs provided a measure of the rate of total protein synthesis within transfected cells. 1 …”

Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells

“…RNase T1 digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis was performed on tRNA Ile UAU as described (18). MALDI peaks were observed corresponding to oligonucleotides present in tRNA Ile UAU but absent in the other Ile isoacceptor, tRNA Ile AAU (UALp, CUCGp, CACCAp, CUPCGp, ACC7DGp, CUPAP6ACGp).…”

“…Dihydrouridines present in bulk or purified isoacceptor tRNAs were reduced with NaBH 4 and labeled with either Rhodamine 110 (Rd110) or Cy3-hydrazide as described (16,18). The labeling efficiencies were 0.87 Rd110/bulk tRNA, 0.86 Rd110/tRNA Ile UAU , 0.68 Rd110/tRNA Gly CCC , 0.68 Cy3/bulk tRNA, 0.83 Cy3/tRNA Ile UAU and 0.48 Cy3/tRNA Pro AGG .…”