Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop

Abstract

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF-Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNAfMet and Cy3-labeled fMetPhe-tRNAPhe, to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3′-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process. Published by Cold Spring Harbor Laboratory Press. Copyright © 2009 RNA Society.