Pyrrolysine Analogues As Substrates for Pyrrolysyl-Trna Synthetase

FEBS Letters 580 (2006) 6695–6700

Pyrrolysine analogues as substrates for pyrrolysyl-tRNA synthetase

Carla R. Polycarpob,1, Stephanie Herringb, Ame´lie Be´rube´a,2, John L. Wooda, Dieter So¨lla,b, Alexandre Ambrogellyb,* a Department of Chemistry, Yale University, New Haven, CT 06520-8114, USA b Department of Molecular Biophysics and Biochemistry, Yale University, P.O. Box 208114, 266 Whitney Avenue, New Haven, CT 06520-8114, USA

Received 24 October 2006; accepted 8 November 2006

Available online 20 November 2006

Edited by Lev Kisselev

assists in the recoding event. Although putative secondary Abstract In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the structures have been identified, their role in recoding in vivo re- mRNAs of some methyltransferases. Pyl is directly acylated mains uncertain [7,8]. onto a suppressor tRNAPyl by pyrrolysyl-tRNA synthetase Determination of the crystal structure of native Methanosar- (PylRS). Due to the lack of a readily available Pyl source, we cina barkeri MtmB allowed the elucidation of the molecular looked for structural analogues that could be aminoacylated by structure of this new amino acid. Analysis of the electron PylRS onto tRNAPyl. We report here the in vitro aminoacylation density showed that pyrrolysine is a dipeptide composed of a Pyl of tRNA by PylRS with two Pyl analogues: N-e-D-prolyl-L- lysine modified at its e-N by a 4-methyl-pyrroline-5-carboxyl- lysine (D-prolyl-lysine) and N-e-cyclopentyloxycarbonyl-L-lysine Pyl ate (Fig. 1) [1]. The electron density of the MtmB crystal struc- (Cyc). Escherichia coli, transformed with the tRNA and ture indicated an anti relationship between the C4-methyl PylRS genes, suppressed a lacZ amber mutant dependent on group and the amide group, and it also suggested the presence the presence of D-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNAPyl of a double bond between the nitrogen and C2 in the pyrroline Pyl and D-prolyl-lysine-tRNA as substrates during protein synthe- ring [1]. sis. Furthermore, the formation of active b-galactosidase shows The detailed characterization of the novel synthetase PylRS that a specialized mRNA motif is not essential for stop-codon will rely on the production of practical amounts of Pyl. Based recoding, unlike for selenocysteine incorporation. on the first reported structure of Pyl, for which the nature of Ó 2006 Federation of European Biochemical Societies. Published the C4 group was not ascertained, we initially undertook the by Elsevier B.V. All rights reserved. synthesis of the Pyl derivative bearing a hydroxyl group at the C4 ring position. As the synthesis of this compound was unsuc- Keywords: Pyrrolysine; Pyrrolysyl-tRNA synthetase; tRNA; cessful, we then devised a new synthetic route leading to the Aminoacyl-tRNA synthetase more stable Pyl derivative bearing a methyl group at the C4 position (see supplemental data). The product obtained (Fig. S1A, compound 8) allowed us to experimentally demon- strate the direct attachment of Pyl to tRNAPyl by PylRS [6]. 1. Introduction Pyl produced by a different synthetic route [9], could be inserted in the M. barkeri MtmB protein in an E. coli context [5]. In the Methanosarcinaceae, a family of methanogenic However, in our hands this synthesis did not yield the desired archaea, the new amino acid pyrrolysine (Pyl) was shown to product. be present in the mono-, di- and tri-methylamine methyltrans- Because these syntheses have a number of drawbacks, chief ferases (MtmB, MtbB and MttB) [1,2]. This new amino acid is among them the complexity of the synthesis, low yields and co-translationally inserted in response to an in-frame UAG high costs, we investigated the possibility of using simpler codon located in the corresponding mRNAs [3]. The insertion and readily available Pyl structural analogues that would still of this amino acid relies on the presence of a specific suppressor be substrates for PylRS and could be inserted into proteins tRNA (tRNAPyl) [4] and the new class II aminoacyl-tRNA in E. coli. synthetase, pyrrolysyl-tRNA synthetase (PylRS) specific only for its substrates tRNAPyl and Pyl [5,6]. The mechanism of Pyl-tRNAPyl insertion at UAG is unknown, however, it has 2. Materials and methods been proposed that a specialized pyrrolysine insertion element (PYLIS) is present immediately downstream of the UAG which 2.1. General Oligonucleotide synthesis and DNA sequencing were performed by the Keck Foundation Biotechnology Resource Laboratory at Yale University. Uniformly labeled sodium [32P] pyrophosphate [1–60 Ci/ *Corresponding author. Fax: +1 203 432 6202. mmol (1 Ci = 37 GBq)], [c-32P] ATP (6000 Ci/mmol) and [3H] serine E-mail address: [email protected] (A. Ambrogelly). (28 Ci/mmol) were from Amersham Biosciences. Mouse raised mono- 1Present address: Instituto de Bioquimica Medica, Universidade clonal anti-E. coli b-galactosidase antibody was purchased from US Federal do Rio de Janeiro, Rio de Janeiro, Brazil. Biological. The pyrrolysine analogues purchase from Sigma were 2 Present address: Department of Chemistry, Saint Joseph College, L-lysine (Fig. 1, compound 1), N-e-methyl-L-lysine (Fig. 1, compound West Hartford, CT, USA. 2), N-e-formyl-L-lysine (Fig. 1, compound 3), N-e-acetyl-L-lysine (Fig. 1, compound 4), and N-e-cyclopentyloxycarbonyl-L-lysine (Cyc) Abbreviations: Pyl, pyrrolysine; PylRS, pyrrolysyl-tRNA synethetase (Fig. 1, compound 5). The pyrrolysine analogues N-e-D-prolyl-L-lysine

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.11.028 6696 C.R. Polycarpo et al. / FEBS Letters 580 (2006) 6695–6700

32 2.3. ATP-[ P] PPi exchange with pyrrolysine analogues The reactions were performed at 37 °C, as described in [11],in 100 mM HEPES-NaOH (pH 7.2), 10 mM MgCl2, 50 mM KCl, 32 5 mM DTT, 2 mM KF, 2 mM ATP, 2 mM [ P] PPi (2 cpm/pmol), 200 nM of PylRS, and 500 lM of a pyrrolysine analogue (Fig. 1, compounds 1–8) in a final volume of 0.1 ml. [32P] ATP formation was followed by specific absorption on acid-washed Norit [0.2 ml of a 1% suspension (wt/vol) of Norit in a solution of 0.4 M sodium pyrophosphate solution containing 15% (vol/vol) perchloric acid], filtration on Whatman GF/C filters, and washing with 15 ml of water and 5 ml of ethanol. Cyc and D-prolyl-lysine concentration were varied from 300 lM to 3 mM for KM determination.

2.4. Aminoacylation assays and acid urea gel electrophoresis of aminoacyl-tRNA Aminoacylation assays were performed for 90 min at 37 °Cin 100 mM HEPES-NaOH (pH 7.2), 30 mM KCl, 15 mM MgCl2, 5 mM ATP, 5 mM DTT, 2 lM tRNAPyl transcript, 1.6 lM PylRS, and 500 lM Cyc, D-prolyl-lysine or PylE. All reactions were started by addition of the amino acid. The aminoacylation level was then visualized by acid urea gel electrophoresis and Northern blot as previously described [6].

2.5. Suppression of E. coli XAC/A24 lacI–lacZ nonsense mutation E. coli strain XAC/A24 carries an inactivating mutation (Trp 220 to UAG nonsense) in the lacI–lacZ fusion system [12]. The truncated protein resulting from premature termination of protein synthesis at the in-frame stop codon is unable to degrade the chromogenic 2-nitro- phenyl b-D-galactopyranoside (ONPG). E. coli strain XAC/A24 cells were co-transformed by electroporation with plasmids carrying the M. barkeri pylS (pCBS) and M. barkeri pylT (pTECH) genes. The same strain carrying an E. coli amber suppressor tRNALys on a pGFIB plasmid [13] was used as a positive control. Transformants were plated on Luria–Bertani (LB) agar with both ampicillin and chloramphenicol and incubated for 16 h at 37 °C. Colonies were transferred to 5 ml of LB liquid medium in the presence of antibiotics and cultured overnight at 37 °C. Fresh LB liquid medium was inoculated with the overnight culture in the presence of antibiotics. Strains carrying the pylS and pylT genes were further supplemented with either 10 mM Cyc, 10 mM D-prolyl-lysine, or no amino acid for control. Cells were culti- vated at 37 °C until A600 reached 0.6. The UAG suppression level was determined by quantitative analysis of the b-galactosidase activity, Fig. 1. Pyrrolysine analogues tested as substrates for M. barkeri which was performed according to the standard procedure [14]. Values are the average of triplicate measurements and are reported as the per- PylRS: L-lysine (1); N-e-methyl-L-lysine (2); N-e-formyl-L-lysine (3); centage of mutant enzyme activity relative to that of the wild type en- N-e-acetyl-L-lysine (4); N-e-cyclopentyloxycarbonyl-L-lysine (Cyc) (5); zyme produced by the E. coli I-Z40 strain, which carries the lacI–lacZ N-e-D-prolyl-L-lysine (D-prolyl-lysine) (6); N-e-L-prolyl-L-lysine (L- fusion with a wild type tryptophan codon in place of the UAG triplet. prolyl-lysine) (7); N-e-(3-methyl-2,3-dihydro-pyrrol-2-yl)carbonyl-L- lysine (PylE) (8); pyrrolysine (Pyl) (9). Compounds 1–3 and 7 could not be activated by PylRS, and compound 4 only showed activity 2.6. Detection of LacI–LacZ fusion protein by immunoblot analysis slightly above background. Compounds Cyc (5), D-prolyl-lysine (6) E. coli XAC/A24 strain carrying the plasmid copy of M. barkeri pylS and PylE (8) were all activated by PylRS and could be charged onto and pylT genes was grown overnight in 5 ml of LB in the presence or tRNAPyl in vitro. absence of 10 mM Cyc. The same strain carrying an E. coli amber suppressor tRNALys on a pGFIB plasmid [13] was used as a positive control. Cell free extracts were prepared, filtered on a Microcon 50 (Millipore), concentrated in a speed-vac, and analyzed on a 4–20% (D-prolyl-lysine) (Fig. 1, compound 6), N-e-L-prolyl-L-lysine (L-prolyl- polyacrylamide/SDS gel (Biorad). The protein extracts were trans- lysine) (Fig. 1, compound 7) and N-e-(3-methyl-2,3-dihydro-pyrrol-2- ferred onto PVDF membrane by using a Bio-Rad blotter. The mem- yl)carbonyl-L-lysine (PylE) (Fig. 1, compound 8) were synthesized as brane was incubated with different dilutions of the anti-E. coli described in the supplemental data section. The M. barkeri Fusaro b-galactosidase antibodies (1:100–1:1000) for optimal detection. For tRNAPyl and PylRS were prepared as previously reported [6]. E. coli the immunoblot analysis, the colorimetric Opti-4CN substrate and strains XAC/A24 and FTP5822 were a gift from H. Nakano (Kyoto detection kit (Bio-Rad) was used (horseradish peroxidase conjugate). University) and E. Murgola (University of Texas), respectively. E. coli I-Z40 stain and of the pGFIB plasmid encoding E. coli suppres- Lys 2.7. Suppression of E. coli FTP5822 trpA nonsense mutation sor tRNA were obtained from W. McClain (University of Wiscon- E. coli strain FTP5822 carries an inactivating mutation in the trpA sin-Madison). gene (Gln 243 to UAG nonsense) [15] which results in tryptophan auxotrophy. Cells were co-transformed by electroporation with plasmids 2.2. Plasmids carrying pylS and pylT genes for in vivo expression carrying M. barkeri pylS (pCBS) and M. barkeri pylT (pTECH) genes. The M. barkeri pylS gene was PCR amplified and cloned into the Transformants were plated on LB agar with both ampicillin and chlo- pCR2.1-TOPO. After sequence confirmation, it was sub-cloned into ramphenicol and incubated for 16 h at 37 °C. Colonies were trans- the pCBS2 vector (Ampr) under the control of the trpS promoter for ferred to 5 ml of LB liquid medium in the presence of antibiotics constitutive expression. The M. barkeri tRNAPyl gene was constructed and cultured for 16 h at 37 °C. Subsequently, cells were washed with from two oligonucleotides inserted between the lpp promoter and the M9 minimal medium and used to inoculate 2 ml of M9 minimal rrn terminator of the chloramphenicol resistance-encoding pTECH medium containing antibiotics and 10 mM Cyc or no amino acid as vector [10]. a control. E. coli strain FTP5822 transformed with pGFIB plasmid C.R. Polycarpo et al. / FEBS Letters 580 (2006) 6695–6700 6697

Lys encoding E. coli amber suppressor tRNA and empty pTECH vector N-e-(3-methyl-2,3-dihydro-pyrrol)oxycarbonyl-L-lysine (PylE) was used as a positive control. Cells were incubated at 37 °C. Bacterial 32 (Fig. 2A). Determination of the KM value in ATP-[ P] PPi growth was followed by measuring cell density over time using a exchange reaction for Cyc (K 670 lM) and D-prolyl-lysine Spectronic 20D+spectrophotometer (Thermo). Values are the average M of triplicate measurements. (KM 500 lM) showed a loss in apparent affinity for PylRS of Measurement of the tryptophan synthetase activity allowed a more about 10-fold when compared to the published value for pyrr- quantitative measurement of the suppression efficiency than growth olysine (KM 53 lMin[5]). In addition to demonstrating amino curves. Overnight cultures of E. coli FTP5822 in M9 minimal medium acid activation, the analogues Cyc, D-prolyl-lysine and PylE were used to inoculate 5 ml of M9 minimal medium, as before, in the Pyl presence or absence of 10 mM Cyc. All cultures were incubated at were successfully ligated onto tRNA by PylRS in vitro 37 °C to late log phase, harvested by centrifugation, washed twice with (Fig. 2B). NaCl (0.9%), and resuspended in buffer A (0.05 M potassium phos- Interestingly, while D-prolyl-lysine was a substrate for PylRS phate (pH 7.0), 0.1 mg/ml of pyridoxal-5-phosphate, 10 mM 2-mercap- the analog L-prolyl-lysine was not, indicating the crucial toethanol). Cell extract was prepared [16], micro-dialyzed against importance of the pyrrolysine ring C-5 stereocenter for recog- buffer A and used immediately for the assay. IGP was freshly prepared as described [8]. Tryptophan synthetase was assayed in the IGP to Trp nition by the synthetase. The importance of C-5 (R) conforma- conversion with [3H] serine (28.0 Ci/mmol) [17]. Enzymatic unit is tion has been suggested before [6]. Our attempt at synthesizing defined as the amount producing 1 lmol of tryptophan/mg of protein, pyrrolysine yielded a mixture of diastereoisomers in which the and expressed as percentage of mutant enzyme activity relative to that stereoconformation of the carbon bearing the amide group of the wild type TrpA in E. coli strain W3110. was not fixed (see supplemental data). Upon analysis of the amino acid attached by the PylRS to tRNAPyl in vitro, we saw that only one of the diastereoisomers was selected by the 3. Results and discussion synthetase [6]. The pyrroline ring nitrogen does not make a strong contribution to PylRS binding since its presence in Pyl 3.1. Pyl analogues are activated and ligated onto tRNA by D-prolyl-lysine only resulted in a small improvement in affinity PylRS in vitro over that of Cyc, but the incorrect positioning of the nitrogen Pyrrolysine is essentially a modified lysine. The crystal struc- in the PylRS active site appears to be detrimental to amino ture of the M. barkeri MtmB enzyme has shown that this novel acid activation. The molecular basis for the rejection of the amino acid is the result of the coupling of a C-4 methyl pyrro- L-prolyl-lysine isomer may become apparent when the struc- lyine ring to the lysine e primary amine via an amide bond [1,9]. tural analysis of the amino acid binding site of PylRS becomes While pyrrolysine is the natural substrate of PylRS, lysine is not available [18]. The need to reject a Pyl isomer with an incorrect recognized by the enzyme [6]. In an attempt to obtain a suitable ring stereoconformation is essential as the UAG-coded posi- substrate for PylRS biochemical characterization and gain tion for Pyl is in the active site of the methyltransferase insight on the contribution of the pyrrolyine ring to pyrrolysine enzymes and presumably essential for enzyme activity [1]. recognition by PylRS, we looked for commercially available or One possible source of a pyrrolysine isomer with the incor- easily synthesizable lysine analogues that would progres- rect conformation may be from the coupling of a member of sively add components of the pyrrolyine ring (Fig. 1). We the proline biosynthetic pathway to lysine, as proline and Pyl then measured the ability of these analogues to promote may share an intermediate metabolite with some structural 32 the PylRS catalyzed ATP-[ P] PPi exchange reaction. N-e- homology. Although nothing is known on Pyl biosynthesis it methyl-L-lysine, N-e-formyl-L-lysine and N-e-L-prolyl-L-lysine has been hypothesized that Pyl might result from the coupling 1 (L-prolyl-lysine) were not able to be activated by PylRS (data of the D-methyl-D -pyrroline-5-carboxylate ring to lysine by an not shown), and N-e-acetyl-L-lysine allowed measurement only amino acid ligase that remains to be identified [19]. Cells con- 1 slightly above background (Fig. 2A). The N-e-cyclopentyl- tain significant concentrations of L-D -pyrroline-5-carboxyl- oxycarbonyl-L-lysine (Cyc) and N-e-D-prolyl-L-lysine (D- ate, an intermediate metabolite in proline biosynthesis and 1 prolyl-lysine) compounds were activated by PylRS, although close structural analogue to D-methyl-D -pyrroline-5-carboxyl- not as efficiently as the pyrrolysine enamine derivative ate. The erroneous coupling of the proline precursor to lysine

Fig. 2. Activation and ligation onto tRNAPyl transcript of pyrrolysine analogues by M. barkeri PylRS. (A) Time course for the activation of pyrrolysine analogues: PylE (d), Cyc (j), L-prolyl-lysine (m), N-e-acetyl-L-lysine (r), no amino acid control (). (B) In vitro aminoacylation of tRNAPyl transcript with Pyl analogues as visualized by acid urea gel electrophoresis: tRNAPyl transcript (L1), tRNAPyl aminoacylated with Cyc (L2), Pyl Pyl tRNA aminoacylated with D-prolyl-lysine (L3), tRNA aminoacylated with PylE (L4), and aminoacylation reaction with no amino acid (L5). 32 Pyl ATP-[ P] PPi exchange reaction and tRNA transcript aminoacylation with the pyrrolysine analogues were conducted as described in Section 2. 6698 C.R. Polycarpo et al. / FEBS Letters 580 (2006) 6695–6700 would then result in a Pyl isomer with an incorrect stereocon- moderately better substrate than Cyc (KM 670 lM) for amino formation at the ring level, and consequently interfere with acid activation by PylRS in vitro, yet the addition of D-prolyl- methyltransferase activity. lysine in the growth medium resulted in slightly lower suppression efficiency relative to growth on Cyc. This suggests either a 3.2. Insertion of Pyl analogues into proteins in E. coli more limited level of cellular uptake of D-prolyl-lysine or cellu- Since Cyc and D-prolyl-lysine were suitable substrates for lar degradation. PylRS in vitro, we then tested whether they would be sub- In addition to quantifying LacI–LacZ activity, we also di- strates in an in vivo context and whether they would be incor- rectly established that UAG read-through in lacI–lacZ mRNA porated into proteins. Previous experiments have shown that was dependent on Cyc using an immunoblot analysis. This when Pyl is added to the growth media of an E. coli strain con- showed the presence of a full length 150 kDa LacI–LacZ pro- taining plasmids bearing pylS, pylT, and mtmB genes from M. tein only in cells grown in the presence of Cyc, implying the barkeri, Pyl can be inserted into MtmB proteins at UAG, indi- incorporation of the Pyl analogue at the suppression site with- cating that the bacterial translation machinery can use Pyl- in lacI (Fig. 3B). Finally, we analyzed the in vivo charging level tRNAPyl [5]. In order to be able to visualize and quantify the of tRNAPyl using the acid gel electrophoresis method. While level of suppression obtained upon insertion of the Pyl ana- tRNAPyl extracted from cells grown in the presence of Cyc logues we used a b-galactosidase based reporter system. The was significantly aminoacylated, it was only present in a deacy- E. coli XAC strains in which a UAG in-frame codon has been lated form when Cyc was absent from the culture media inserted in the lacI part of a lacI–lacZ fusion gene have been (Fig. 3C). Combined, this evidence indicates PylRS is specifi- extensively used to assess sequence-function relationships of cally acylating tRNAPyl with the Pyl analogs in vivo, and the a wide range of tRNA molecules by quantifying the b-galacto- E. coli translation machinery is able to use this charged tRNA sidase activity resulting from suppression of the amber stop co- in protein synthesis at levels comparable to other amber sup- don [20–22]. We transformed the E. coli XAC/A24 strain with pressor aminoacyl-tRNAs. plasmids carrying pylS and pylT genes. The b-galactosidase In order to see whether Cyc insertion is being influenced by activity could only be detected when the transformant cells the particular context in which the UAG codon is placed in were grown in LB containing either Cyc or D-prolyl-lysine, lacI–lacZ, we investigated whether the Pyl analogue could be indicating that the PylS/tRNAPyl pair is truly orthogonal in incorporated at a similar level in another E. coli protein. E. coli and that the Pyl analogues are inserted in the LacI– Because extensive analysis of E. coli trpA mutant strains have LacZ fusion protein (Fig. 3A). The b-galactosidase activity been conducted previously [15], and used as a powerful tool to in cells grown with the Pyl analogues was compared to that screen libraries of mutant tRNAs [23], we chose to use this sys- from either an E. coli strain containing in its genome a wild tem as a reporter for Cyc incorporation. The E. coli strain type copy of the lacI–lacZ fusion gene or from the E. coli FTP5822 carries an inactivating mutation in the trpA gene XAC/A24 strain transformed with a plasmid carrying an (Gln 243 to UAG nonsense), which results in a tryptophan E. coli amber suppressor tRNALys. The amber suppressor auxotrophy phenotype due to the organism’s inability to syn- tRNALys allowed 38% read-through efficiency as previously thesize this amino acid. This phenotype can be rescued either reported [13], and the suppression efficiency in cells grown when the cells are grown on an exogenic source of tryptophan on Cyc and D-prolyl-lysine were 25% and 18%, respectively or when provided with a suitable suppressor tRNA. We trans- (Fig. 3A). Interestingly, D-prolyl-lysine (KM 500 lM) was a formed the E. coli FTP5822 strain with plasmids carrying the

Fig. 3. Incorporation of Cyc and D-prolyl-lysine into b-galactosidase. (A) b-Galactosidase activity catalyzed by the wild type enzyme produced by the E. coli I-Z40 strain, which carries the lacI–lacZ fusion with a wild type tryptophan codon in place of the UAG triplet (L1), E. coli XAC/A24 carrying lacI–lacZ with Trp220 to UAG mutation transformed with E. coli suppressor tRNALys (L2), E. coli XAC/A24 strain transformed with M. barkeri pylS and pylT grown in presence of either 10 mM Cyc (L3) of 10 mM D-prolyl-lysine (L4), no amino acid (L5), E. coli XAC/A24 strain transformed with pylT and empty pCBS2 vector (L6). (B) In vivo aminoacylation of tRNAPyl measured by acid gel electrophoresis. Total tRNA extracted from E. coli XAC/A24 strain transformed with M. barkeri pylS and pylT grown in presence (L3) or absence (L5) of 10 mM Cyc. (C) Immunoblot of full-length LacI–LacZ fusion protein. Total protein extract from E. coli XAC/A24 strain transformed E. coli suppressor tRNALys (L2) and from E. coli XAC/A24 strain transformed with M. barkeri pylS and pylT grown in presence (L3) or absence (L5) of 10 mM Cyc. C.R. Polycarpo et al. / FEBS Letters 580 (2006) 6695–6700 6699

Fig. 4. Incorporation of Cyc and D-prolyl-lysine into tryptophan synthase. (A) Growth curve in M9 minimal media of E. coli FTP5822 strain transformed with E. coli suppressor tRNALys (j), E. coli FTP5822 strain transformed with M. barkeri pylS and pylT grown in presence (d)or absence (s) of 10 mM Cyc, E. coli FTP5822 strain transformed with either M. barkeri pylS (h)orM. barkeri pylT (n) and grown in presence of 10 mM Cyc. (B) In vivo aminoacylation of tRNAPyl as judged by acid gel electrophoresis. Total tRNA extracted from E. coli FTP5822 strain transformed with M. barkeri pylS and pylT grown in presence (L1) or absence (L2) of 10 mM Cyc.

pylS and pylT genes and grew the cells in M9 minimal media in tion of the Pyl required for active site characterization of the presence or absence of Cyc. The presence of the Pyl PylRS. Furthermore, we establish that the PLYIS element is analogue in the culture media restored bacterial growth, imply- not essential for pyrrolysine insertion at UAG codons, but raise ing that a functional full-length tryptophan synthase had been the possibility that PYLIS may enhance read-through levels. made (Fig. 4A). This was confirmed by assaying tryptophan synthase activity in cell free extracts of the E. coli grown in Acknowledgements: We are grateful to Frances Pagel, Emanuel Mur- presence of Cyc (2.6 U/mg, 16% of wild type activity). To com- gola, Hiroaki Nakano and William McClain for the gift of plasmids and E. coli strains. This work was supported NIH grant GM22854 pare suppression activity, E. coli FTP5822 strain was also (to D.S.), and by support from Bristol-Myers Squibb, Amgen, Yama- Lys transformed with the amber suppressor tRNA and trypto- nouchi, Pfizer, and Merck (to J.L.W.). phan synthase activity was measured (5.5 U/mg, 33% of wild type activity). As with the lacI–lacZ read-through system, tRNAPyl suppression was reduced relative to the suppressor Appendix A. Supplementary data tRNALys, but still significant. Analysis of the in vivo charging level of tRNAPyl by the acid gel electrophoresis method Supplementary data associated with this article can be showed aminoacylation of tRNAPyl only when cells were found, in the online version, at doi:10.1016/j.febslet.2006. grown in presence of Cyc (Fig. 4B). 11.028. Previous studies of pyrrolysine insertion at UAG in E. coli examined read-through in the mtmB gene which contains the PYLIS element structure [5]. Here, we show that tRNAPyl References charged with D-prolyl-lysine and Cyc can be inserted at [1] Hao, B., Gong, W., Ferguson, T.K., James, C.M., Krzycki, J.A. UAG in both lacZ and trpA mRNA which presumably lack and Chan, M.K. (2002) A new UAG-encoded residue in the any PYLIS element. These results establish that a PYLIS structure of a methanogen methyltransferase. Science 296, 1462– element is not essential for signaling pyrrolysine insertion at 1466. UAG. This is in contrast to selenocysteine insertion, which is [2] Soares, J.A., Zhang, L., Pitsch, R.L., Kleinholz, N.M., Jones, R.B., Wolff, J.J., Amster, J., Green-Church, K.B. and Krzycki, dependent on the presence of a selenocysteine insertion J.A. (2005) The residue mass of L-pyrrolysine in three distinct element (SECIS). It is possible the PYLIS, while not absolutely methylamine methyltransferases. J. Biol. Chem. 280, 36962– required, is important for enhancing the suppression level in 36969. the methyltransferase gene. [3] James, C.M., Ferguson, T.K., Leykam, J.F. and Krzycki, J.A. (2001) The amber codon in the gene encoding the monomethyl- amine methyltransferase isolated from Methanosarcina barkeri is 3.3. Conclusions and perspectives translated as a sense codon. J. Biol. Chem. 276, 34252– We identified two Pyl analogues, the commercially available 34258. N-e-cyclopentyloxycarbonyl-L-lysine (Cyc) and N-e-D-prolyl- [4] Srinivasan, G., James, C.M. and Krzycki, J.A. (2002) Pyrrolysine encoded by UAG in Archaea: charging of a UAG-decoding L-lysine (D-prolyl-lysine) which is easily synthesized, that can specialized tRNA. Science 296, 1459–1462. circumvent the lack of a reliable and cheap source of synthetic [5] Blight, S.K., Larue, R.C., Mahapatra, A., Longstaff, D.G., Pyl for both in vitro and in vivo study of PylRS. By doing so, Chang, E., Zhao, G., Kang, P.T., Green-Church, K.B., Chan, we also established the essentiality of the pyrroline ring stereo- M.K. and Krzycki, J.A. (2004) Direct charging of tRNACUA with chemistry, thus gaining some preliminary insight on the stru- pyrrolysine in vitro and in vivo. Nature 431, 333–335. [6] Polycarpo, C., Ambrogelly, A., Be´rube´, A., Winbush, S.M., cture–activity relationships for Pyl recognition by PylRS. McCloskey, J.A., Crain, P.F., Wood, J.L. and So¨ll, D. (2004) An Determination of the chemical pathway to Pyl and the genes aminoacyl-tRNA synthetase that specifically activates pyrroly- responsible for Pyl biosynthesis will allow the future produc- sine. Proc. Natl. Acad. Sci. USA 101, 12450–12454. 6700 C.R. Polycarpo et al. / FEBS Letters 580 (2006) 6695–6700

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