Author:
Kaur Jaskiran,Raj Monika,Cooperman Barry S.
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 NaBH4, 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 matrix-assisted 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 tRNAPhe and E. coli tRNAArg. 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 NaBH4 reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA.
Publisher
Cold Spring Harbor Laboratory
Cited by
29 articles.
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