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.
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Footnotes
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↵1 Present address: Department of Biological Chemistry and Molecular Pharmacology, Harvard University School of Medicine, Boston, MA 02115, USA.
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Reprint requests: Barry S. Cooperman, University of Pennsylvania, Department of Chemistry, Philadelphia, PA 19104-6323, USA; e-mail: cooprman@pobox.upenn.edu; fax: (215) 898-2037.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1257509.
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- Received July 7, 2008.
- Accepted October 24, 2008.
- Copyright © 2009 RNA Society
