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. 2008 Oct;14(10):2245–2253. doi: 10.1261/rna.1158508

Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3′ end

Chun-Mei Zhang 1, Cuiping Liu 1, Thomas Christian 1, Howard Gamper 1, Jef Rozenski 2, Dongli Pan 3, John B Randolph 4, Eric Wickstrom 1, Barry S Cooperman 3, Ya-Ming Hou 1
PMCID: PMC2553749  PMID: 18755841

Abstract

All mature tRNA molecules have the conserved CCA sequence at the 3′ end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 3′ end. Using Escherichia coli tRNACys as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNACys, separated from the unlabeled precursor tRNA by reverse phase high-pressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNACys with E. coli CysRS reveals an equilibrium K d closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyC-labeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.

Keywords: pyrrolo-cytosine ribonucleoside, aminoacylation, ribosome translocation, tRNACys, tRNA CCA adding enzyme, fluorescence

INTRODUCTION

The universally conserved CCA sequence is located at positions 74–76 of all mature tRNA molecules and protrudes as a single-stranded segment from the helical acceptor stem. This sequence is an essential element in the process of decoding genetic information. It provides the terminal A76 ribose for amino acid attachment to tRNA catalyzed by aminoacyl-tRNA synthetases (aaRSs) (Ibba et al. 2005). Some aaRSs cannot sufficiently discriminate against incorrect amino acids and thus possess a separate editing site that hydrolyzes incorrectly synthesized aminoacyl-tRNA (aa-tRNAs) (Freist 1989). A correctly matched aa-tRNA enters the ribosome A site at a codon position that forms correct base pairings with the anticodon of the tRNA. While on the ribosome, the CCA sequence forms specific Watson–Crick base pairs with conserved residues of the rRNA in the large ribosomal subunit (Samaha et al. 1995; Nissen et al. 2000; Korostelev et al. 2006; Selmer et al. 2006). These interactions stabilize tRNA in the A and P sites to enable ribosome-catalyzed peptide bond formation and promote stable translocation of tRNA (Yusupova et al. 2006) in each elongation cycle of protein synthesis.

Crystal structures of tRNA in complexes with components of the ribosomal translational machinery reveal that the CCA sequence is dynamic and can adopt multiple conformations. For example, class I aaRS enzymes typically bend the CCA sequence of their cognate tRNA into a hairpin to access the active site (e.g., Rould et al. 1989), whereas class II enzymes maintain the CCA sequence in a straight conformation (e.g., Ruff et al. 1991). In the editing site, however, class I aaRS enzymes flip the hairpin conformation of CCA into a straight conformation (e.g., Silvian et al. 1999), whereas class II aaRS enzymes fold the CCA sequence into a hairpin (e.g., Dock-Bregeon et al. 2004). Additional examples of CCA mobility are provided by the changes in conformation within tRNA–aaRS complexes that are induced by addition of cognate amino acid (Sherlin and Perona 2003) or by correct aaRS recognition of the anticodon loop (Hauenstein et al. 2004). Similarly, chemical tRNA footprinting (Moazed and Noller 1989a, b), kinetic (Dorner et al. 2006; Pan et al. 2007), and FRET (Ermolenko et al. 2007) studies have provided evidence that the CCA ends of A and P site tRNAs move into hybrid states during the elongation cycle of protein synthesis, in which the CCA ends of tRNAs move toward the P and E sites on the large subunit, while the anticodons of tRNAs remain in A and P sites on the small subunit.

The diverse conformational possibilities of the CCA end suggest that the development of a molecular probe that can directly monitor the conformational mobility of the CCA sequence would be highly useful. An earlier study (Ott et al. 1989) incorporated a fluorophore at position 75 in tRNATyr and tRNAPhe, using the CCA enzyme ((ATP(CTP): tRNA nucleotidyl trasnferase) that catalyzes step-wise nucleotide addition to positions 74, 75, and 76 (Yue et al. 1996). In this case, the tRNA was synthesized to C74 and was extended by the CCA enzyme with the 2-thiocytidine (S2C) analog to position 75 and the normal A to position 76. The S2C analog was then alkylated with the fluorophore N-iodoacetyl-N′-(5-sulfo-1-naphthyl) ethylenediamine (1,5-I-AEDANS). However, because the AEDANS fluorophore is bulky, the fluorescent tRNA prepared as such was not a substrate for aminoacylation, preventing its direct application to reactions that require charged aa-tRNA.

To overcome the previous limitations, we have recently developed a method of fluorescent labeling of the tRNA 3′ end, replacing AEDANS with the less bulky fluorescent cytosine analog pyrrolo-C (PyC). The PyC analog was previously shown to be an excellent probe for protein–nucleic acid interactions (Berry et al. 2004; Tinsley and Walter 2006), because it can be selectively excited at 350 nm, well separated from the absorbance at 260 nm of natural nucleotides, and has an emission maximum of 460 nm, far removed from that of protein tryptophans (emission=340 nm). In addition, its fluorescence is sensitive to local conformational changes of nucleic acids, which has been exploited to study the dynamics and stabilities of DNA helices, RNA duplexes, and a DNA/RNA hybrid (Liu and Martin 2002; Tinsley and Walter 2006). Our method of fluorescent labeling of tRNA uses the CCA enzyme to incorporate PyC and generates fluorescent products that are substrates for aminoacylation. The enzymatic incorporation is cost effective as compared to the cost of incorporation of PyC by chemical synthesis for the full-length tRNA. We recently applied our labeling method to the 3′ end of Methanococcus jannaschii tRNACys to examine the activities of the PyC-labeled tRNA in the indirect pathway for synthesis of Cys-tRNACys (Zhang et al. 2008). In this pathway the tRNA is first phosphoserylated by O-phosphoseryl-tRNA synthetase (SepRS) to form Sep-tRNACys, which is then converted to Cys-tRNACys by Sep-tRNA–Cys-tRNA synthase (SepCysS) (Sauerwald et al. 2005). We describe here the details of the PyC-labeling method of the tRNA 3′ end and show that PyC-labeled tRNA is also highly active on the ribosome.

RESULTS AND DISCUSSION

Incorporation of PyC to position 75 in the CCA sequence

Although the CCA-adding enzyme uses CTP and ATP as the normal nucleotide substrates, a large variety of base analogs of the natural substrates are functional as well. For example, the Bacillus stearothermophilus (Bst) CCA-adding enzyme recognizes base analogs that retain the imine N3 as the hydrogen-bond (H-bond) acceptor and exocyclic N4 of C as the H-bond donor (Cho et al. 2003). Because PyC maintains these functional groups as in the normal C (Fig. 1A), it was tested as a substrate analog for the Bst CCA-adding enzyme, which was purified as a His-tag fusion from an overexpression clone in Escherichia coli (Cho et al. 2003).

FIGURE 1.

FIGURE 1.

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PyC as a fluorescence probe in E. coli tRNACys. (A) The chemical structure of PyCTP used for incorporation by Bst CCA-adding enzyme. Inward and outward arrows denote hydrogen bond acceptors and donors, respectively. (B) A scheme showing the cloverleaf of E. coli tRNACys-C74 as the substrate for Bst CCA enzyme for incorporation of PyC75 and A76, using PyCTP and ATP as the substrates.

We chose E. coli tRNACys as a sequence framework different than that of M. jannaschii tRNACys to test the enzymatic incorporation of PyC by the Bst CCA enzyme. E. coli tRNACys was transcribed from the first nucleotide to C74 to allow the CCA enzyme to incorporate a single PyC into position 75 (Fig. 1B). The DNA template for transcription, including the promoter sequence for T7 RNA polymerase, is constructed from overlapping oligonucleotides and contains C2′-O-methyl ribose at the first two positions at the 5′ end of the template strand. These modifications reduce the heterogeneity of nucleotide addition at the tRNA 3′ end (Kao et al. 1999). The quality of the transcript (designated as tRNA-C74) was verified by the following control experiments. First, the tRNA-C74 transcript was body labeled with 32P during transcription and purified from a denaturing 12% acrylamide/7 M urea gel. It migrated as a homogeneous band in an analytical denaturing gel (Fig. 2A, lane 1). Second, incubation of the purified transcript, heat-denatured and reannealed in 10 mM MgCl2, with the Bst CCA enzyme and ATP showed no extension of the transcript (Fig. 2A, lane 2), whereas incubation with both ATP and CTP gave extension to position 76 (Fig. 2A, lane 4). The dependence on both ATP and CTP for extension to position 76 confirmed that the transcript indeed terminated at position 74. Third, incubation of the transcript with CTP (but no ATP) resulted in two extension products, one to position 75 and the other to position 76 (Fig. 2A, lane 3). These products are, respectively, tRNA-C74C75 and tRNA-C74C75C76 (where the incorporated residues are underlined). The synthesis of the latter product is consistent with the polyC activity of CCA enzymes, when CTP is used as the sole nucleotide substrate (Hou 2000; Seth et al. 2002; Hou et al. 2005).

FIGURE 2.

FIGURE 2.

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Incorporation of PyC75 to E. coli tRNACys. (A) A denaturing gel analysis (12% PAGE/7 M urea) of incorporation of PyC75 to the transcript of E. coli tRNACys. The final concentration of each component is indicated in parentheses. The full-length tRNA-A76 and the truncated tRNA-C74 were body labeled by transcription with α-32P-ATP, which provided a marker on the left. The body-labeled tRNA-C74 was used as the substrate for reactions 1–6, whereas the unlabeled tRNA-C74 was used with α-32P-ATP for reaction 7. The symbols “+” and “−” indicate the presence and absence of a reagent, respectively. The denaturing gel was run in 90 mM Tris-HCl (pH 8.3), 90 mM boric acid, and 2 mM EDTA at 1800 V for 2 h at 60°C. (B) RP-HPLC chromatogram of the CCA reaction on the C8 column developed by increasing concentration of methanol (shown in blue as percent in v/v). The elution profile of tRNA-C74 before the CCA reaction is shown in black, while that after the reaction is shown in red. (C) RP-HPLC chromatogram of the CysRS aminoacylation reaction of tRNA-CPyCA generated from extension of tRNA-C74 on the C8 column developed by increasing concentration of methanol (shown in blue as percent in v/v).

PyCTP (Glen Research) was evaluated as a substrate for the Bst CCA enzyme for incorporation into position 75 in E. coli tRNACys. In the presence of PyCTP only, tRNA-C74 was extended by the CCA enzyme to position 76 (Fig. 2A, lane 5), indicating recognition of the analog by the enzyme and synthesis of tRNA-C74PyC75PyC76. Previous studies have shown that the incorporation of C76 by CCA enzymes is suppressed upon the addition of ATP so that A76 is preferentially incorporated (Hou 2000; Seth et al. 2002). This was tested in a reaction that included both PyCTP and ATP, which led to extension of tRNA-C74 to position 76 (Fig. 2A, lane 6). The identity of the product was evaluated by using [α-32P]ATP as the substrate and unlabeled tRNA-C74 as the primer. The extension product, which migrated to the same position as the full-length tRNA (Fig. 2A, lane 7), was labeled. Because PyC was present in a large excess of the labeled ATP, the incorporation of the label suggests preferential synthesis of tRNA-C74PyC75A76.

To further characterize the enzymatic synthesis of tRNA-CPyCA, the extension reaction of tRNA-C74 by Bst CCA enzyme in the presence of PyCTP and ATP was scaled up from that shown in lane 6 of Figure 2A. The reaction product was injected to a reverse phase HPLC (RP-HPLC) C8 column, and the chromatogram was developed by a linear gradient of methanol (Fig. 2B). The peak with retention time of 12 min was designated as the tRNA-C74 primer due to its presence both before and after the CCA reaction, while the peak with retention time of 16 min was designated as the tRNA-CPyCA product due to its presence only in the sample after the CCA reaction. The incomplete conversion of tRNA-C74 to tRNA-CPyCA in this case, in contrast to the complete conversion as shown in Figure 2A, was due to a twofold reduction in the molar ratio of the Bst CCA enzyme to the tRNA substrate in the scaled-up reaction. The material in the tRNA-CPyCA peak was a substrate for aminoacylation by E. coli CysRS, indicating that it possessed the correct 3′ end sequence. For example, the tRNA product extended by Bst CCA enzyme was subjected to aminoacylation by E. coli CysRS in the presence of cysteine and ATP. RP-HPLC analysis of the aminoacylation reaction showed a 50% reduction of the tRNA-CPyCA peak concomitant with the appearance of a new peak with retention time of 8 min (Fig. 2C). When 35S-cysteine was used in the aminoacylation reaction, the radioactivity coeluted with the new peak, indicating that the new peak corresponded to the charged Cys-tRNA-CPyCA.

The material in the tRNA-CPyCA peak was digested to mono-nucleosides by nuclease P1 and alkaline phosphatase, followed by analysis using liquid chromatography coupled to mass spectrometry. A peak containing PyC was clearly identified in the liquid chromatogram (Fig. 3A) exhibiting the correct mass-to-charge ratios (m/z) of 282 for the protonated nucleoside and m/z of 150 for the protonated base fragment (Fig. 3B). Peaks of all natural nucleosides were clearly identified as well. The RP-HPLC-purified tRNA-CPyCA (Fig. 2B) and Cys-tRNA-CPyCA (Fig. 2C) were employed in the biochemical assays described below.

FIGURE 3.

FIGURE 3.

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Liquid chromatography/mass spectrometry analysis of the E. coli tRNACys-CPyCA transcript digest to single nucleosides. (A) RP-chromatogram on C18 showing the UV trace at 260 nm and the percent (v/v) of acetonitrile. The commercially available PyCTP (Glen Research) digested to the nucleoside form was used to provide the marker for PyC with the retention time of 20.5 min. (B) Mass spectrometry of the PyC peak isolated from A, showing the m/z value of 282.1 for the protonated nucleoside of PyC and m/z value of 150.1 for the protonated PyC base.

Aminoacylation of E. coli tRNACys-CPyCA

In saturating assay conditions, purified E. coli tRNACys-CPyCA was aminoacylated to 40% by E. coli CysRS, which is approximately twofold lower than the 70% level achieved with the unlabeled tRNA-CCA (Fig. 4A). This is consistent with ∼50% reduction of the tRNA-CPyCA peak upon aminoacylation, giving rise to the charged Cys-tRNACys as presented in Figure 2C. Additionally, purified tRNACys-CPyCA exhibited similar kinetics of aminoacylation as the unlabeled tRNACys transcript. A representative aminoacylation reaction is shown in Figure 4B, where the PyC-labeled tRNA showed a k cat/K m of 0.36 ± 0.05 μM−1s−1 for aminoacylation (determined at a tRNA concentration <10% of the K m for E. coli CysRS), which is approximately twofold reduced from those of the unlabeled tRNACys (Fig. 4B). Two methods were used to prepare the unlabeled tRNA: tRNACys-CCA was synthesized by extension of the tRNA-C74 transcript to A76, in parallel with the procedure used to synthesize tRNACys-CPyCA, whereas tRNACys-A76 was synthesized by transcription to A76. Kinetic analysis under the same conditions gave an identical k cat/K m value (0.8 ± 0.1 μM−1 s−1) for each. These values agree well with those reported previously (Christian et al. 2000; Zhang et al. 2003b; Zhang and Hou 2005). Based on the stoichiometries and catalytic efficiencies of aminoacylation presented in Figure 4, PyC75 in the CCA sequence interferes with the E. coli CysRS aminoacylation activity to some extent, which is expected because the modification is immediately adjacent to the site of aminoacylation at A76. However, the extent of interference is minor, suggesting that PyC75 is a suitable probe for examining the interaction of the tRNA 3′ end with tRNA synthetases.

FIGURE 4.

FIGURE 4.

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Aminoacylation of tRNA by E. coli CysRS. (A) Aminoacylation plateau of 1 μM of tRNA-CCA and tRNA-CPyCA with 35S-cysteine catalyzed by 4 μM of CysRS. The percent of aminoacylation was based on the amount of synthesized 35S-cysteinyl-tRNACys versus that of the input tRNA as determined from absorption at 260 nm (1 OD260 = 40 μg/mL). (B) Aminoacylation kinetics of 1 μM of tRNA-A76, tRNA-CCA, and tRNA-CPyCA (as purified from Fig. 2B) with 35S-cysteine catalyzed by 1 nM of CysRS. The concentration of tRNA was determined from absorption at 260 nm while that of CysRS was determined from the Bradford assay and corrected by active site titration.

Equilibrium binding of the CysRS-tRNACys complex

PyC75 was tested for its utility to monitor the equilibrium binding interaction in the E. coli CysRS-tRNACys complex. As shown in the cocrystal structure of the complex, the CCA end enters into the ATP-binding site in the absence of ATP or an analog (Hauenstein et al. 2004). Thus, the binding interaction was monitored in the presence of saturating concentrations of cysteine and the nonhydrolyzable AMPcPP (where the α- and β-phosphates are bridged by a CH2 group). The fluorescence emission spectrum of the PyC-labeled tRNA exhibited the expected maximum at 460 nm. Upon binding E. coli CysRS, the fluorescence spectrum of PyC showed a small blue shift (from 460 to 448 nm) and a marked increase in emission (1.7-fold measured at λmax) (Fig. 5A). A titration of the PyC fluorescence increase as a function of CysRS concentration was fit to a hyperbolic equation, which revealed a K d of 2.2 ± 0.2 μM (Fig. 5B). Previously, the intrinsic tryptophan fluorescence of E. coli CysRS had been used to determine the enzyme affinity for tRNACys in the presence of CysAMS (5′-O-[N-(L-cysteinyl) sulfamoyl] adenosine), which is an analog of the Cys-AMP intermediate of the aminoacylation reaction. The K d value determined from this study was 1.9 ± 0.1 μM (Hauenstein et al. 2004), closely similar to the K d value determined by using PyC as a probe. The similarity in K d values validates the use of the PyC probe in measuring the CysRS-tRNACys interaction. In contrast, titration of the PyC-labeled tRNACys with the noncognate E. coli AlaRS in the presence of alanine and AMPcPP showed no change in fluorescence up to 13 μM of the enzyme (Fig. 5C). The lack of a fluorescence response to AlaRS demonstrates that the PyC readout in the tRNACys-CysRS interaction was synthetase specific.

FIGURE 5.

FIGURE 5.

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Fluorescence of PyC-labeled E. coli tRNACys. (A) Emission spectrum of E. coli tRNACys-CPyCA (0.1 μM) in the presence of cysteine (0.5 mM) and AMPcPP (1 mM), with and without E. coli CysRS (13 μM). (B) Replot of fluorescence increase of the tRNA upon binding E. coli CysRS as a function of the enzyme concentration and fit to a hyperbolic binding equation to derive the K d of 2.2 ± 0.2 μM. (C) Emission spectrum of E. coli tRNACys-CPyCA (0.1 μM) in the presence of alanine (0.5 mM) and AMPcPP (1 mM), with and without E. coli AlaRS (13 μM).

Earlier work based on the intrinsic tryptophan fluorescence of E. coli CysRS showed that a truncated enzyme mutant containing just the anticodon-binding domain exhibited the same affinity for tRNA (K d = 0.3 ± 0.1 μM) as the full-length enzyme (Zhang and Hou 2005). This suggests that tRNA binding to CysRS in the absence of cysteine or an ATP analog is dominated by enzyme interaction with the tRNA anticodon. Indeed, when PyC75 was used to probe the tRNA acceptor end interaction with CysRS, no fluorescence change was observed up to an enzyme concentration of 100 μM (data not shown). The lack of PyC fluorescence change indicates little binding interaction between the CCA end and the enzyme active site and/or no major alterations of the acceptor end region of the enzyme–tRNA binary complex. This result, together with the change of K d from the binary complex (K d = 0.3 ± 0.1 μM) to the ternary complex (K d = 2.2 ± 0.2 μM), is consistent with the notion that formation of the ternary complex leads to structural rearrangements that bring the 3′ end of the tRNA into close contact with CysRS, as had been inferred from structural analysis (Hauenstein et al. 2004). Thus, comparison of the PyC fluorescence, which monitors the CCA sequence, with the synthetase intrinsic fluorescence, which monitors the entire tRNA molecule, sheds new light on the conformational rearrangement of the synthetase–tRNA complex during aminoacylation.

Kinetics of translocation of PyC-labeled tRNACys on the ribosome

Translocation of tRNA on the ribosome is catalyzed by the factor EF-G in a GTP bound form. Because translocation involves the most extensive movements of tRNA in each elongation cycle of protein synthesis (Yusupov et al. 2001; Stark et al. 2002; Valle et al. 2002; Konevega et al. 2007), investigation of the kinetics of translocation is important for understanding the fidelity of decoding. The kinetics of tRNA translocation have been monitored by fluorescence changes in proflavin (prf) or rhodamine incorporated into the D-loop of the tRNA tertiary core (Wintermeyer and Zachau 1979; Robertson and Wintermeyer 1981; Paulsen and Wintermeyer 1986; Robertson et al. 1986; Rodnina et al. 1994, 1997; Pan et al. 2006; Betteridge et al. 2007). It has been demonstrated that translocation of peptidyl-tRNAPhe (prf) from the A site in the pretranslocation complex (PRE) to the P site in the post-translocation complex (POST) proceeds via a two-step reaction, in which a rapidly formed metastable intermediate is more slowly converted to the POST complex (Pan et al. 2007). This intermediate exhibited a puromycin reactivity that fell in between those of PRE and POST complexes (Pan et al. 2007).

To test the utility of PyC as a probe for monitoring tRNA translocation and to determine whether an intermediate is formed by a tRNA different from tRNAPhe, the PyC-labeled E. coli tRNACys was tested for translocation from the A to P site. A PRE complex containing PyC-labeled fMet-Cys-tRNACys in the A site and tRNAfMet in the P site was formed and rapidly mixed with 1 μM of the EF-G-GTP complex, giving rise to fluorescence changes that were monitored in a stopped-flow spectrophotometer. The time course (Fig. 6) showed a two-phase reaction, with an initial increase in fluorescence intensity, proceeding with an apparent rate constant of 19.0 ± 1.7 s−1, followed by a decrease of fluorescence intensity, proceeding with an apparent rate constant of 3.6 ± 2.0 s−1. The corresponding values for translocation of fMet-Phe-tRNAPhe (prf), also measured at 1 μM of EF-G, are 9 ± 1 s−1 and 3.4 ± 0.3 s−1 (Pan et al. 2007). The observation of the two-phase translocation kinetics with comparable rate constants between the two tRNAs clearly demonstrates the utility of the PyC probe for monitoring tRNA movement on the ribosome. However, because the two tRNAs differ in their sequence framework and in the placement of the fluorophore, direct comparison of their rate constants is not possible. Nonetheless, the twofold faster rate of intermediate formation of PyC-labeled tRNACys might arise from (1) small differences between the intrinsic structures of Cys-tRNACys and Phe-tRNAPhe; (2) the lack of post-transcriptional modification in the tRNACys transcript used in these experiments, as compared to the native tRNAPhe used previously (Pan et al. 2007); and/or (3) differential interactions with the ribosome made by PyC at position 75 and by proflavin in the tRNA D loop.

FIGURE 6.

FIGURE 6.

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Translocation kinetics. PyC-labeled E. coli Cys-tRNACys (2 μM) was loaded onto the A site of the programmed E. coli ribosome (1 μM) and rapidly mixed with EF-G-GTP (1 μM). The fluorescence change of the translocation time course of the tRNA from A to P site is shown, displaying the rapid formation of an intermediate (k 1 = 19.0 ± 1.7 s−1) from the PRE complex and a slower conversion of the intermediate to the POST complex (k 2 = 3.6 ± 2.0 s−1).

Conclusion

We have demonstrated that PyC can be incorporated by the Bst CCA enzyme to E. coli tRNACys, in addition to M. jannaschii tRNACys, as a probe to monitor the activities and dynamics of the tRNA 3′ end. The fluorescent E. coli tRNACys-CPyCA contains PyC, as confirmed by mass spectrometry, and exhibits the same gel electrophoretic mobility as normal tRNA. In addition, it is functional for aminoacylation and for ribosomal translocation, two tRNA activities that depend on the integrity of the 3′ end. Thus, although we have not directly compared the enzymatically synthesized PyC-labeled tRNA with a chemically synthesized counterpart, the enzymatically synthesized tRNA displays the correct biochemical features. This enzymatic fluorescent labeling of the tRNA 3′ end is easy to implement and is applicable to all tRNA sequence frameworks. Notably, PyC functions as the nucleotide substrate for the CCA enzyme for incorporation after C74, and once incorporated, it also functions as the primer for A76 addition. We focus on position 75 to limit the PyC incorporation to one well-defined position. In certain cases, it may be beneficial to incorporate PyC at both positions 74 and 75 in order to enhance the fluorescence signal of PyC. This can be achieved by preparing a tRNA primer that terminates at position 73. However, due to the rapid reaction of CCA enzymes to synthesize consecutive C74 and C75, it would be difficult to incorporate PyC to just position 74. Importantly, we emphasize that the placement of the PyC label at the CCA sequence provides a new tool to monitor the kinetics and thermodynamics of the labeled tRNA for protein binding interactions to yield potentially new information that might be quite distinct from the readout of previous studies based on enzyme intrinsic tryptophan fluorescence or on fluorescent probes placed at other regions of tRNA. Such distinctions should provide new insights into the dynamics of protein–tRNA interactions. Studies currently underway will further exploit the potential of the PyC labeling approach described in this work.

MATERIALS AND METHODS

Preparation of tRNA and enzymes

Transcripts of E. coli tRNACys, ending at C74 and A76, respectively, were prepared by run-off transcription by T7 RNA polymerase, using the BstN1-restricted pTFMaCys01 plasmid as the template for the tRNA ending at A76 and a pair of overlapping oligonucleotides as the template for the tRNA ending at C74. The two overlapping oligonucleotides are designed with the ribose 2′-O-methyl moiety placed at the 5′ terminus on the DNA template strand (Kao et al. 1999). Transcripts of the correct lengths were separated from aborted sequences by electrophoresis in a 12% polyacrylamide gel with 7 M urea (PAGE/7 M urea), identified by UV shadowing, excised from the gel, and crushed and soaked in TE (10 mM Tris-HCl at pH 8.0, 1 mM EDTA), followed by ethanol precipitation. The concentrations of the transcripts were determined based on absorption at 260 nm (1 OD260 = 40 μg/mL). E. coli CysRS with a C-terminal His-tag was expressed from the pET-22 vector as described (Zhang et al. 2003a). The overexpression clone of Bst CCA-adding enzyme with a C-terminal His-tag was a gift of Dr. Alan Weiner (University of Washington) and the enzyme was purified as described (Cho et al. 2003).

Incorporation of PyC75 by the Bst CCA-adding enzyme

PyCTP was provided by Glen Research. Extension of the tRNA-C74 transcript of E. coli tRNACys (32P-labeled, 10 μM) was performed in 100 mM glycine-NaOH (pH 9.0), 10 mM MgCl2, 1 mM DTT, with 1 mM ATP, 0.5–1 mM CTP or PyCTP, and 0.1–0.5 μM Bst CCA enzyme. After incubation at 60°C for 30 min, extension was terminated with 7 M urea and products were resolved by a 12% PAGE/7 M urea and purified. The extension reaction can be scaled up to 500 μL with 1 mM ATP, 1 mM CTP, 20 μM tRNA, and 0.5 μM enzyme.

Mass spectrometry analysis of the incorporated PyC75 nucleotide

The E. coli tRNACys-CPyCA transcript (800 pmol) was digested to nucleosides by nuclease P1 and shrimp alkaline phosphatase. The final volume of this reaction mixture was 10 μL, of which 0.25 μL were injected directly onto the HPLC system (CapLC; Waters). The separation of the nucleosides was performed on a reversed phase column (Alltima HP C18 AQ, 150 mm × 300 μm; Alltech) thermostatted at 40°C. Acetonitrile (HPLC gradient grade; Fisher Scientific) 84% in water was used as organic phase and ammonium formate 25 mM (pH 6.3) as aqueous phase. The flow rate was 4.5 μL/min and the gradient started at 2% organic phase. The components were eluted by increasing the organic phase by 1%/min during 25 min. Electrospray mass spectra were recorded in positive ionization mode on an orthogonal acceleration quadrupole time-of-flight mass spectrometer (Q-Tof 2; Micromass), which was coupled to the HPLC system. Electrospray capillary voltage was set to +3000 V. Mass spectra were recorded every 2 sec in the m/z range 100–350. The applied collision cell energy (12 eV) caused a partial fragmentation of the protonated nucleosides [M+H]+, revealing the protonated base peak [BH+H]+.

Aminoacylation of tRNACys by E. coli CysRS

Aminoacylation with 35S-cysteine (12,000 dpm/pmol) was performed with 0.2–1 μM tRNA and 1 nM CysRS for kinetic analysis, but 4 μM CysRS for determination of aminoacylation plateau. Aliquots of aminoacylation reactions were terminated by the carboxymethylation reaction and spotted on filter pads and precipitated by 5% TCA as described (Zhang et al. 2003a,b). To isolate the Cys-tRNACys product, a saturating concentration of the unlabeled cysteine (500 μM) was used, with 5 mM ATP and 20–30 μM tRNACys, and incubated with 4 μM CysRS at 37°C for 5 min. The reaction was terminated with the addition of 0.3 M sodium acetate (pH 6.0), extracted by phenol (pH 6.0), and ethanol precipitated.

RP-HPLC

Products of the Bst CCA reaction and the E. coli CysRS aminoacylation reactions were applied to a semi-analytical C8 column (Alltima, 0.5 × 25 cm, pore diameter 300 Å, particle diameter 3 μm, manufactured by Alltech). An HPLC system (SCL-10AVP; Shimadzu) was equilibrated with buffer A (20 mM ammonium acetate at pH 5.5, 50 mM NaCl) and eluted with increasing concentration of buffer B (60% methanol in buffer A). Eluates were recovered by ethanol precipitation and assayed for aminoacylation.

Fluorescence measurements

Fluorescence of E. coli tRNACys-CPyCA (0.1 μM) in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 5 mM DTT, 0.5 mM cysteine, and 1 mM AMPcPP was monitored on a bench top QM-4 fluorometer (Photon Technology International). The excitation wavelength was set at 352 nm, while the emission was recorded from 400 to 600 nm. Addition of E. coli CysRS enhanced the emission intensity, and the concentration-dependent increase in intensity at 450 nm was used to calculate the percentage of fluorescence change. The background signal, provided from a buffer control, was subtracted from each spectrum. The fluorescence change vs enzyme concentration was fit to the hyperbolic equation: y = ([E] × F max)/([E] + K d) by Sigma plot, where [E] is the enzyme concentration and F max is the maximum fluorescence change.

Translocation of tRNA on the ribosome

Tight coupled 70S ribosome, IF1, IF2, IF3, EF-Tu, EF-G, and fMet-tRNAfMet were made or obtained as described (Pan et al. 2006, 2007). The mRNA sequence was 5′-GGGAAGGAGGUAAAAAUGUGCAAACGUAAAUCUACU-3′, where the underlined AUG was the start codon and the UGC triplet immediately after was the codon for tRNACys. The following experiments were performed in buffer: 50 mM Tris-HCl (pH 7.5), 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2, and 1 mM DTT. Initiation complex with fMet-tRNAfMet on the P site was formed by incubating 1 μM ribosomes with 1.5 μM IF1, 1.5 μM IF2, 1.5 μM IF3, 1.5 μM fMet-tRNAfMet, 4 μM mRNA, and 1 mM GTP at 37°C for 25 min. Ternary complex was formed by incubating 4 μM EF-Tu, 1 mM GTP, and 2 μM pyrrolo-Cys-tRNACys, purified by RP-HPLC as in Figure 2C, at 37°C for 5 min. The two complexes were then mixed and incubated at 37°C for 30 sec to form the PRE complex. Pretranslocation complex (0.1 μM final concentration) was rapidly mixed with EF-G.GTP (1 μM final concentration) on a SX.18 MV stopped-flow spectrofluorometer at 25°C. Time courses were fit to the equation y = y0 + A1 exp(k 1*t) + A2 exp(k 2*t) by Igor-Pro (Wavemetrics).

ACKNOWLEDGMENTS

We thank Alan Weiner for providing the Bst CCA adding enzyme clone and Georges Lahoud for assistance with figures. This work was supported by grants from NIH (GM66267 to Y.-M.H., GM71014 to B.S.C.) and from DOE/BER (ER63055 to E.W.).

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.1158508.

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