Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop.
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 tRNA(fMet) and Cy3-labeled fMetPhe-tRNA(Phe), 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.
Pan
Dongli
D
Department of Chemistry, University of Pennsylvania, Philadelphia, 19104-6323, USA.
Qin
Haiou
H
Cooperman
Barry S
BS
D002232:Carbocyanines / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D008722:Methods; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000138:chemical synthesis / Q000737:chemistry; D012343:RNA, Transfer / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D054679:Ribosome Subunits, Small / Q000378:metabolism; D014529:Uridine / Q000737:chemistry*
D002232:Carbocyanines; D005456:Fluorescent Dyes; D010455:Peptides; D012331:RNA, Fungal; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C089187:cyanine dye 3; C085321:cyanine dye 5; C089238:fMet-tRNA(fMet); D011072:Poly U; C012001:polyphenylalanine; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D014529:Uridine