Fluorescent labeling of tRNAs for dynamics experiments

Abstract

Transfer RNAs (tRNAs) are substrates for complex enzymes, such as aminoacyl-tRNA synthetases and ribosomes, and play an essential role in translation of genetic information into protein sequences. Here we describe a general method for labeling tRNAs with fluorescent dyes, so that the activities and dynamics of the labeled tRNAs can be directly monitored by fluorescence during the ribosomal decoding process. This method makes use of the previously reported fluorescent labeling of natural tRNAs at dihydrouridine (D) positions, but extends the previous method to synthetic tRNAs by preparing tRNA transcripts and introducing D residues into transcripts with the yeast enzyme Dus1p dihydrouridine synthase. Using the unmodified transcript of Escherichia coli tRNAPro as an example, which has U17 and U17a in the D loop, we show that Dus1p catalyzes conversion of one of these Us (mostly U17a) to D, and that the modified tRNA can be labeled with the fluorophores proflavin and rhodamine 110, with overall labeling yields comparable to those obtained with the native yeast tRNAPhe. Further, the transcript of yeast tRNAPhe, modified by Dus1p and labeled with proflavin, translocates on the ribosome at a rate similar to that of the proflavin-labeled native yeast tRNAPhe. These results demonstrate that synthetic tRNA transcripts, which may be designed to contain mutations not found in nature, can be labeled and studied. Such labeled tRNAs should have broad utility in research that involves studies of tRNA maturation, aminoacylation, and tRNA-ribosome interactions.

FIGURE 1.

Reaction scheme for two-step fluorescent labeling of tRNA. In step 1, a tRNA transcript is synthesized by transcription and modified by Dus1p. In step 2, the Dus1p-modified tRNA transcript is reduced by NaBH₄ in alkali to open the nonaromatic dihydrouracil ring, following by replacement of the ureidopropanal group with a primary amine-bearing fluorophore, such as rhodamine 110.

FIGURE 2.

(A) Sequence and cloverleaf structure of E. coli tRNA^Pro/UGG. The primer binding site for reverse transcription (residues G22–G42) complementary is indicated by the curved line with an arrow. (B) Primer extension products analyzed by 12% PAGE/7 M urea. M indicates the position of the starting primer, which was 5′ end labeled. Reactions 1 and 2 contained 0.6 and 1.2 μg of E. coli tRNA^Pro/UGG modified by Dus1p, respectively, while reactions 3 and 4 contained the unmodified transcript.

FIGURE 3.

Determination of the D content in tRNA by the colorimetric assay. (A) Proportionality of A550 to increasing concentration of E. coli bulk tRNA or the transcript of E. coli tRNA^Pro. The slope of each linear relationship is converted to D/tRNA according to the calibration of A ₅₅₀ from titration of increasing concentration of dihydrouracil. (B) Average of D/tRNA value from five independent determinations as shown in (A), graphed for the bulk E. coli tRNA and the transcript of E. coli tRNA^Pro.

FIGURE 4.

Separation of fluorescent-labeled tRNA from RNase H-mediated cleavage of unlabeled tRNA by gel electrophoresis. Transcripts of (A) yeast tRNA^Phe and (B) E. coli tRNA^Pro were prepared, with and without the rhodamine label, and were hybridized to an appropriate oligonucleotide complementary to the region of the label. After hybridization, these transcripts were subjected to cleavage by RNase H according to (Hou et al. 2006) and were analyzed by a 12% PAGE/7 M urea. The unlabeled transcript of yeast tRNA^Phe was cleaved by RNase H to generate fragments of 61-mer and 15-mer, respectively, while the unlabeled transcript of E. coli tRNA^Pro was cleaved to generate fragments of 60-mer and 17-mer, respectively. The labeled transcript of each tRNA was resistant to the cleavage and was isolated as a full-length tRNA from the gel.

FIGURE 5.

(A) Emission spectra of native yeast tRNA^Phe modified with rhodamine at positions 16 and 17 and the purified transcript of E. coli tRNA^Pro/UGG modified with rhodamine at position 17a. (B) Single-turnover kinetics of translocation with k _app of 4.61 ± 0.04 s⁻¹ for the native yeast tRNA^Phe(prf) and k _app of 3.50 ± 0.04 s⁻¹ for the transcript of yeast tRNA^Phe(prf), monitored by stopped-flow fluorescence.