ANALYTICALBIOCHEMISTRY

Analytical Biochemistry 335 (2004) 192–195

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A method for coupled transcription and aminoacylation of cysteinyl-tRNA

Ioana Pavela, Angela Belcherb, Karen S. Browninga,¤

a Department of Chemistry Biochemistry, Institute for Cell and Molecular Biology, and Center for Nano- and Molecular Science and Technology, University of Texas at Austin, Austin, TX 78712, USA

b Division of Biological Engineering, Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Received 25 May 2004

Abstract

A novel method for coupled transcription and aminoacylation of transfer RNA was developed where Escherichia coli cysteine-speciWc tRNA (tRNAcys) was transcribed and aminoacylated in a single reaction. The cys-tRNAcys that was synthesized andaminoacylated using this method was functional in in vitro translation. The cys-tRNAcys was further modiWed with biotin (N-iodo-acetyl-N-biotinhexylenediamine) to facilitate detection. The biotin-modiWed cys-tRNAscys was also functional in in vitro translation,allowing the synthesis and detection of biotin-labeled protein. 2004 Elsevier Inc. All rights reserved.

Large-scale in vitro synthesis of a speciWc RNA ismade possible using T7, SP6, or T3 RNA promoters andpolymerases [1]. This technique has been used successfullyto transcribe RNAs having a variety of lengths [2–4]. Invitro transcribed tRNAs may be aminoacylated [2,5–7]and used in translation [3,4]. Biotinylated aminoacylatedtRNAs have also been used successfully in in vitro trans-lation [8], and the use of nonradioactive translation sys-tems has become an important biochemical tool [8–16].

In this article, we describe a novel method for thesimultaneous transcription and aminoacylation, or cou-pled transcription-aminoacylation (CTA),1 of tRNAcys

using a single-step reaction. This new method is time-

* Corresponding author. Fax: +1 512 471 8696.E-mail address: kbrowning@mail.utexas.edu (K.S. Browning).

1 Abbreviations used: CTA, coupled transcription-aminoacylation;cys-tRNAcys, cysteinyl-tRNAcys; E. coli Hisc-CysRS, Escherichia coli cys-teinyl-tRNA synthetase; UTP, uridine-5�-triphosphate; Ni-NTA, nickel-nitrilotriacetic acid resin; ILCB, N-iodoacetyl-N-biotinhexylenediamine;GFP, green Xuorescent protein; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PVDF, polyvinylideneXuoride.

0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2004.08.029

eYcient, eliminating the need to purify the tRNA priorto aminoacylation, and produces a cysteinyl-tRNAcys

(cys-tRNAcys) that is functional in in vitro translation.The cys-tRNAcys prepared by CTA can be further modi-Wed with a biotin group and used successfully to synthe-size proteins in vitro that may be detected by the biotinfunctional group.

Materials and methods

Plasmids containing the genes for tRNAcys [5] andEscherichia coli cysteinyl-tRNA synthetase (E. coli Hisc-CysRS) [17] were a gift from Y-M. Hou (Thomas JeVer-son University, Philadelphia, PA, USA). Expression andpuriWcation of the 6£ His tag labeled E. coli Hisc-CysRSwas carried out as described previously [17].

Coupled transcription–aminoacylation

The plasmid containing the tRNAcys gene with a T7promoter was digested with BstNI to linearize the

I. Pavel et al. / Analytical Biochemistry 335 (2004) 192–195 193

template for transcription. Digestion with BstNI leaves atemplate for the 3� CCA end of the transcribed tRNA.T7-Megashortscript transcription kits (Ambion, Austin,TX, USA) were used according to the manufacturer’sinstructions. The digested template (0.36–0.50 mg/ml)was transcribed in a 100-�l reaction mixture volume. Tocouple the transcription and charging reactions, themixture was supplemented with 6–12 �g puriWed E. colicys-tRNAcys synthetase [17] and either 75 mM �-[32P]uri-dine-5�-triphosphate (UTP) (54 dpm/pmol, Perkin–Elmer, Boston, MA, USA) to monitor tRNA formationor 1.2 mM L-[35S]cysteine (1538 dpm/pmol) to monitortRNA aminoacylation. The reaction was incubated at37 °C for 2 h, and aliquots were taken at various timepoints and analyzed by electrophoresis on a 6% urea–acrylamide gel or by precipitation with 10% trichloro-acetic acid [3] or 0.24 M iodoacetic acid/0.1 M sodiumacetate (pH 5.0) (CAM) and 10% trichloroacetic acid [5].

Larger scale synthesis of cys-tRNAcys (0.5 ml) wascarried out as described above and supplemented witheither [35S]cysteine or unlabeled cysteine. E. coli Hisc-CysRS was removed batchwise from the charged tRNAwith nickel-nitrilotriacetic acid resin (Ni-NTA, Qiagen,Valencia, CA, USA). The Ni-NTA matrix (200 �l) waswashed twice in a 1.5-ml microcentrifuge tube with500 �l of 20 mM Hepes–KOH (pH 7.6). The resin wasallowed to settle, and the supernatant was removed.The transcription reaction mixture was added to thematrix and gently mixed by rotation for 1 h at 4 °C. Themixture was then centrifuged for 1 min to pellet theresin, and the supernatant containing the cys-tRNAcys

was removed. This process was repeated once toremove any remaining synthetase. The cys-tRNAcys waspuriWed by gel Wltration on a Sephadex G-25 column asdescribed previously [12]. The eluates containing cys-tRNAcys were pooled (»0.5 ml), lyophilized, and dis-solved in 75 �l of H2O. The yield from a 0.5-ml reactionwas approximately 1.0–1.5 mg. No phenol extraction,ethanol precipitation, or heating/reannealing wascarried out.

Attachment of biotin to cys-tRNA

The attachment of biotin (N-iodoacetyl-N-biotin-hexylenediamine, ILCB) (Pierce Biotechnology, Rock-ford, IL, USA) to [35S]cys-tRNA was carried outaccording to the manufacturer’s instructions. BrieXy,[35S]cys-tRNAcys (21 nmol) was incubated in the darkwith ILCB (37.5 nmol) for 2 h at room temperature. Thebiotinylated cys-tRNAcys was puriWed by Sephadex G-25 column chromatography as described previously [12]and was lyophilized. The eYciency of the biotinylationreaction was estimated with an EZ Biotin QuantitationKit (Pierce Biotechnology) according to the manufac-turer’s instructions. The amount of biotin was approxi-mately 0.25–0.40 pmol biotin/pmol tRNA.

Translation reactions

The E. coli T7 S30 Extract System (Promega, Madi-son, WI, USA) was used for translation assays. PlasmidpIVEX 2.1 encoding green Xuorescent protein (GFP,Roche Applied Science, Indianapolis, IN, USA) wasused as the template in the coupled transcription–trans-lation reaction. The reaction mixture was supplementedwith 2 mM L-[35S]cysteine (1538 dpm/pmol), 2 mM L-[35S]cys-tRNAcys (1538 dpm/pmol), or 2 mM ILCB[35S]cys-tRNAcys (1538 dpm/pmol). The biosynthesis of[35S]GFP was conWrmed by SDS–PAGE and autoradi-ography. The biosynthesis of biotinylated GFP was con-Wrmed by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS–PAGE), blotting to a polyvinylid-eneXuoride (PVDF) membrane, and detection withstreptavidin–horseradish peroxidase conjugate (1/10,000dilution, Pierce Biotechnology) as well as a chemilumi-nescent substrate used according to the manufacturer’sinstructions (Super Signal, Pierce Biotechnology).

Results

The time course of cysteine-speciWc tRNAcys synthesisin an in vitro transcription and a CTA reaction is shownin Fig. 1. To quantitate the amount of synthesized

Fig. 1. Time course of in vitro synthesis of [32P]tRNAcys (transcription)and cys-[32P]tRNAcys (CTA). (A) Aliquots of 2.5 �l were removed atindicated times and mixed with an equal amount of 10£ urea–gel dye.The samples were loaded on a 6% urea–acrylamide gel. The gel wasstained with 0.1% Stains-All (Sigma, St. Louis, MO, USA) and dried.(B) The gel shown in panel A was exposed to a phosphorimager screen(Amersham Biosciences, Piscataway, NJ) to visualize the incorpora-tion of �-[32P]UTP (54 dpm/pmol) into cysteine-speciWc tRNAcys

(transcription) and cys-tRNAcys (CTA). (C) Time course of incorpora-tion of �-[32P]UTP (54 dpm/pmol) into cysteine-speciWc tRNAcys (�)and cys-tRNAcys (�). Aliquots of 5 �l were removed at the indicatedtimes, precipitated with cold 10% trichloroacetic acid, and collected onglass Wber Wlters by vacuum Wltration [3]. The Wlters were dried andcounted in a scintillation counter (Beckman, Fullerton, CA, USA),and the amount of tRNA synthesized was calculated.

194 I. Pavel et al. / Analytical Biochemistry 335 (2004) 192–195

tRNAcys, radiolabeled �-[32P]UTP was added to thereaction mixture (Fig. 1B). Transcription product inboth reaction mixtures increased over the time course;however, transcription in the CTA reaction mixture wasreduced by approximately 25% at later time points (Fig.1C). This may be due to consumption of ATP duringaminoacylation, reducing transcription eYciency. Theratio of transcript (7500 pmol) to template (5.5 pmol)was approximately 1300:1.

The aminoacylation of transcribed tRNAcys was mon-itored by the incorporation of [35S]cysteine. The reactionmixture shown in Fig. 2A contained [35S]cysteine, and theamount of [35S]cysteine incorporated into the tRNAcys isshown in the autoradiogram of the same gel (Fig. 2B).The amount of [35S]cysteine incorporated into cys-tRNA-cys increases with time (as shown in Fig. 2C). A compari-son of the graphs in Fig. 1C (transcription) and Fig. 2B(aminoacylation) shows that both reactions follow a sim-ilar trend. The eYciency of aminoacylation shown in Fig.2 at 2 h was estimated at 10%.

Fig. 2. Time course of in vitro synthesis of [35S]cys-tRNAcys. (A) Aliqu-ots of 2.5 �l were removed at indicated times and mixed with an equalamount of 10£ urea–gel dye. The samples were loaded on a 6% urea–acrylamide gel. The gel was stained with 0.1% Stains-All and dried. (B)The gel in panel A was exposed to a phosphorimager screen to visual-ize the [35S]cysteine (1538 dpm/pmol) incorporation at indicated times.(C) Aliquots of 5 �l were removed at the indicated times and incubatedfor 30 min with 24 �l CAM [5]. The aliquots were precipitated withcold 10% trichloroacetic acid and collected on a glass Wber Wlter byvacuum Wltration. The Wlters were dried and counted in a scintillationcounter. (D) In this phosphorimage of [35S]cys-tRNAcys (300 pmol and1000 cpm/pmol) and ILCB [35S]cys-tRNAcys, lane 1 shows 300 pmol[35S]cys-tRNAcys before reaction with biotin, and lane 2 shows300 pmol [35S]cys-tRNAcys after reaction with biotin ILCB. An aliquotof 2.5 �l was mixed with an equal amount of 10£ urea–gel dye. Thesamples were loaded on a 6% urea–acrylamide gel.

The cys-tRNAcys was coupled to a biotinylated linker,ILCB, to make a biotin-modiWed cys-tRNAcys (ILCB[35S]cys-tRNAcys). Degradation or loss of [35S]cysteinefrom the tRNA during the coupling process was notdetected (cf. lanes 1 and 2 in Fig. 2D).

Both [35S]cys-tRNAcys and ILCB [35S]cys-tRNAcys

were used successfully to synthesize GFP in a coupledtranscription–translation reaction (Fig. 3). The incorpo-ration of [35S]cysteine into GFP was monitored by SDS–PAGE and autoradiography (lane 2 in Fig. 3A and lanes1 and 3 in Wrst panel of Fig. 3B). The incorporation ofILCB cysteine into GFP was monitored by detection ofthe biotin with streptavidin–horseradish peroxidase(lane 3 in second panel of Fig. 3B).

Discussion

A novel method of coupled transcription aminoacyla-tion (CTA) of tRNA molecules was developed andtested using E. coli tRNAcys and its cognate synthetase.The ability to further modify the tRNA with a biotiny-lated probe was also demonstrated. The tRNA synthe-sized by CTA or further modiWed with biotin was shownto be functional in in vitro translation.

Fig. 3. In vitro translation using tRNAs synthesized by CTA. (A) Lane1 shows translation mixture containing [35S]cys-tRNAcys minus GFPplasmid template, and lane 2 shows translation mixture containing[35S]cys-tRNAcys with GFP plasmid template (4 �g). [35S]Cysteineincorporation into GFP was visualized with a phosphorimager. (B) Inboth panels, lane 1 shows translation mixture containing [35S]cys-tRNAcys with GFP plasmid template (4 �g), lane 2 shows translationmixture containing ILCB [35S]cys-tRNAcys minus GFP plasmid tem-plate, and lane 3 shows translation mixture containing ILCB [35S]cys-tRNAcys with GFP plasmid template (4 �g). The phosphorimagershows incorporation of [35S]cysteine into GFP. The biotin detectionassay shows incorporation of ILCB [35S]cysteine into GFP.

I. Pavel et al. / Analytical Biochemistry 335 (2004) 192–195 195

The data show that the tRNAcys was being aminoacy-lated over the course of the transcription reaction (Figs.1B, 2C). This suggests that the tRNAcys is folded in thecorrect form and immediately recognized by the synthe-tase so as to be aminoacylated. A comparison of theresults from transcription and aminoacylation in Figs. 1and 2 shows that the eYciency of aminoacylation was esti-mated at 10%. In separate CTA experiments, the eYciencywas as high as 36% (data not shown). These results sug-gest that the transcription reaction does not generate100% full-length tRNAcys or that some portion is notfolded correctly. T7 polymerase is known to prematurelyterminate transcription or add untemplated nucleotides[18]. Methods to improve Wdelity of the 3�-end formationmay improve the eYciency of aminoacylation.

The coupling of cys-tRNAcys with a biotinylatedlinker, ILCB, produced a biotin-modiWed cys-tRNAcys.The iodoacetyl linkage was chosen for its reactivity tothe sulfhydryl group of cysteine, which yields a thioetherthat is stable in the reducing environment of the transla-tion reaction. The amount of biotin attached to thetRNA (»0.25–0.40 pmol/pmol tRNA) was consistentwith the estimated percentage charging (»10–36%).

The CTA method is a single-step reaction in which thetRNA is transcribed and aminoacylated in the same invitro reaction mixture. This method is eYcient and elimi-nates the need to purify the tRNA prior to aminoacyla-tion. Furthermore, the cys-tRNAcys may be modiWed witheasily detectable functional groups (e.g., biotin) and stillremain functional. Both [35S]cys-tRNAcys and ILCB[35S]cys-tRNAcys were used successfully to synthesize GFPin a coupled transcription–translation reaction (Fig. 3). Nophenol extraction, ethanol precipitation, or reannealingwas performed on the tRNA, allowing for a very time-eYcient “single pot” synthesis of aminoacylated tRNA.

Acknowledgments

We thank Dr. Ya-Ming Hou for the plasmids, and wealso acknowledge Drs. Esther Ryan and Leah Allen fortheir useful comments and input in editing the manu-script. This research was funded by the NSF NanoscaleInterdisciplinary Research Team (NIPT0103473) to A.B.and K.S.P.

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