FLUORESCENT LABELING OF TRANSFER RNA AND STUDY OF PROTEIN SYNTHESIS

摘要

Provided are methods for labeling transfer RNA comprising replacing the u racil component of a dihydrouridine of said transfer RNA with a fluorophore. The disclosed methods may comprise fluorescent labeling of natural tRNAs (i .e., tRNAs that have been synthesized in a cell, for example, in a bacterium , a yeast cell, or a vertebrate cell) at dihydrouridine (D) positions, or fl uorescent labeling of synthetic tRNAs. In another aspect, the present invent ion provides methods for assessing protein synthesis in a translation system comprise providing a tRNA having a fluorophore substitution for the uracil component of a dihydrouridine in a D loop of the tRNA; introducing the label ed tRNA into the translation system; irradiating the translation system with electromagnetic radiation, thereby generating a fluorescence signal from th e fluorophore; detecting the fluorescence signal; and, correlating the fluor escence signal to one or more characteristics of the protein synthesis in th e translation system. The disclosed methods are useful in single molecule as well as in ensemble settings.