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Published in final edited form as: FEBS Lett. 2010 May 21;584(13):2857–2861. doi: 10.1016/j.febslet.2010.05.028

A tRNA-dependent cysteine biosynthesis enzyme recognizes the selenocysteine-specific tRNA in Escherichia coli

Jing Yuan 1, Michael J Hohn 1, R Lynn Sherrer 1, Sotiria Palioura 1, Dan Su 1, Dieter Söll 1,2,*
PMCID: PMC2893348  NIHMSID: NIHMS214368  PMID: 20493852

Abstract

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNACys (Sep-tRNACys) into Cys-tRNACys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase (SepSecS) converts O-phosphoseryl-tRNASec (Sep-tRNASec) to selenocysteinyl-tRNASec (Sec-tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that SeptRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNASec to Cys-tRNASec, and that Sep is crucial for SepCysS recognition.

Keywords: aminoacyl-tRNA, formate dehydrogenase, selenocysteine, O-phosphoseryl-tRNASec kinase, Sep-tRNA:Cys-tRNA synthase, Sep-tRNA:Sec-tRNA synthase

1. Introduction

The tRNA-dependent amino acid modification reactions provide aminoacyl-tRNAs for at least four amino acids [1]. The enzymes involved in this process must recognize the amino acid of the aminoacyl-tRNA as well as a part of the tRNA. Because of the importance of these reactions for protein synthesis, and their possible application for making unnatural aminoacyl-tRNA species, it is desirable to know what part(s) of the tRNA is recognized by these enzymes. There is a reasonable amount of information on the tRNA-dependent amidotransferases, which are responsible for glutaminyl- and asparaginyl-tRNA formation, and also SepSecS, the enzyme that forms Sec-tRNA from Sep-tRNA [2,3].

SepSecS appears to be highly specific for Sep-tRNASec based on the crystal structure of the SepSecS:tRNASec binary complex. Its unique homotetrameric quaternary state not only interacts with the Sep moiety but also specifically recognizes tRNASec by measuring the 13 bp long acceptor/TΨC helix (Fig. 1). The shorter 12 bp acceptor/TΨC helix of canonical tRNAs precludes them from being substrates for SepSecS since the tip of their acceptor arm cannot reach the active site of the enzyme [4]. SepSecS uses selenophosphate as the selenium donor, but cannot differentiate it from its sulfur-containing analog thiophosphate and forms Cys-tRNASec in vitro [4]. This nondiscriminative nature of SepSecS towards thiophosphate and selenophosphate is compensated in vivo by the highly specific activity of selenophosphate synthetase, which discriminates against sulfide and only forms selenophosphate in the presence of ATP [5]. Furthermore, the long acceptor/TΨC helix precludes binding of Sec-tRNASec to EF-Tu and serves as the distinct feature that the selenocysteine-specific elongation factor SelB is recognizing [6,7], which ensures accurate Sec incorporation in response to Sec UGA codons.

Fig. 1. Secondary structures of tRNASec and tRNACys.

Fig. 1

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The cloverleaf structures of E. coli tRNASec (A) and tRNACys (B) are shown.

Similar information on substrate specificity is lacking for SepCysS, the essential enzyme in methanogenic tRNA-dependent cysteine biosynthesis. SepCysS converts Sep-tRNACys to Cys-tRNACys in the presence of a sulfur donor [8,9]. Cys-tRNACys, the product of this tRNA-dependent pathway, is either used for protein synthesis or it provides free cysteine for other biosynthetic pathways via its deacylation [8,9].

SepCysS resembles SepSecS in several aspects. Both enzymes use phosphoserylated tRNAs as substrates and catalyze amino acid conversions by a pyridoxal phosphate (PLP)-dependent mechanism [4,10,11]. The reactions start with the formation of a Schiff base between the phosphoserine moiety of the Sep-tRNA and PLP. This ultimately leads to release of Sep's phosphate group and formation of a PLP-bound dehydroalanyl-tRNA intermediate. Nucleophilic attack of this intermediate by the incoming sulfur or selenium atom yields an oxidized form of Cys- or Sec-tRNA respectively that is subsequently reduced and released from the enzyme. In contrast to SepSecS, little is known about the substrate specificity of SepCysS besides that it can use multiple sulfur donors in vitro such as sulfide, thiophosphate and cysteine [11]. Given the apparent similarity of SepCysS and SepSecS catalysis, the different structures of tRNASec and tRNACys (Fig. 1), and the established in vivo functional assay for tRNASec utilizing formate dehydrogenase [4,10,12,13], we decided to test in vivo whether Sep-tRNASec is a substrate for SepCysS.

2. Material and methods

2.1 General

DNA sequencing was performed by the Keck Foundation Biotechnology Resource Laboratory at Yale University. [75Se]selenite was purchased from the University of Missouri Research Reactor Facility (Columbia, MO).

2.2 Plasmids for in vivo complementation

The plasmids were constructed as described before [12]. Specifically, the PSTK gene (pstK) from Methanocaldococcus jannaschii and selD from E. coli were cloned into the pACYC vectors individually. E. coli selA, M. jannaschii SepSecS and SepCysS genes (encoded by spcS and pscS, respectively) were cloned into pET15b vectors individually.

2.3 Construction of the E. coli ΔselA ΔselD double deletion strain

Construction of the ΔselA ΔselD double deletion strain was carried out according to a published method [14]. The KmR cassette, which is disrupting selA in E. coli strain JS1, was excised by FLP recombinase-mediated homologous recombination between the FRT sites flanking the KmR cassette, upon transformation of the JS1 strain with plasmid pCP20 [14]. In the resulting strain, the selD gene was then disrupted by a FRT-KmR cassette as previously described [14], thus yielding strain MH1.

2.4 Complementation test using the benzyl viologen assay

The E. coli JS1 and MH1 strains were transformed with genes as indicated in the figure legends and tested for FDHH activity by the benzyl viologen assay as described before [4,10,12,13,15]. The transformants were grown anaerobically on glucose-minimum medium agar plates at 30°C for 24-48 h in the presence of 0.01 mM IPTG. After they were removed from the anaerobic jar, the plates were immediately overlaid with 0.75% top agar (containing 1 mg/ml benzyl viologen, 0.25 M sodium formate and 25 mM KH2PO4, pH 7.0).

2.5 Complementation test using the McConkey nitrate assay

E. coli JS1 transformants were plated on McConkey nitrate plates (40 g/L McConkey agar base, 20 g/L KNO3, 1 g/L glycerol, 0.5 g/L sodium formate and 0.1 g/L glucose) [16] and grown anaerobically at 37°C for 24 h.

2.6 Metabolic labeling with radioactive selenium

The procedure was carried out as described [12]. Overnight cultures of JS1 transformants were diluted (1:50) in 5 ml of TGYEP medium (0.5% glucose, 1% tryptone, 0.5% yeast extract, 1.2% K2HPO4, 0.3% KH2PO4, 0.1% formate, 1 μM Na2MoO4, pH 6.5) [17] supplemented with 1 μCi [75Se]selenite and 0.05 mM IPTG, and grown under anaerobic conditions at 37°C for 24 h. The cells were harvested and the cell lysates analyzed by SDS-PAGE followed by autoradiography. Various conditions were used to increase the sensitivity of the assay including a higher amount of [75Se]selenite (10 μCi), higher concentration of IPTG (up to 0.5 mM) and longer anaerobic growth time (up to 48 h).

3. Results

3.1 The tRNASec is a substrate for SepCysS

To test whether SepCysS can utilize Sep-tRNASec as a substrate in vivo, we transformed the E. coli ΔselA strain JS1 with the SepCysS gene (pscS) in the presence or absence of the PSTK gene (pstK). Transformants were then tested for the activities of two selenoproteins, formate dehydrogenase H (FDHH) and formate dehydrogenase N (FDHN) [18]. Active FDHH can use formate to reduce benzyl viologen which results in purple colored colonies [15]. Transformants grown anaerobically on minimal medium plates supplemented with selenite were layered with top agar containing formate and benzyl viologen. Complementation of the selA deletion was observed when strain JS1 was either cotransformed with the PSTK and SepSecS genes, or with E. coli selA (Fig. 2 left). Cells cotransformed with SepCysS and PSTK genes also turned purple, however to a lesser extent, suggesting a reduced FDHH activity (Fig. 2 left). Transformation of the SepCysS gene or PSTK gene alone did not restore FDHH activity in the E. coli JS1 strain (Fig. 2 left).

Fig. 2. SepCysS and PSTK complement an E. coli ΔselA deletion.

Fig. 2

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The indicated proteins (middle) complement the loss of selenocysteine synthase (SelA) in the E. coli ΔselA deletion strain JS1. Activity of the selenoproteins FDHH and FDHN was tested with the benzyl viologen assay (left) and the McConkey nitrate plate assay (right) respectively.

To further confirm these results we tested the transformed JS1 strains for the activity of another selenoprotein, FDHN, which uses nitrate as an electron acceptor when formate is oxidized. Transformants were grown anaerobically on McConkey plates containing nitrate and a pH indicator. Cells containing active FDHN consume nitrate, resulting in an increase of the pH shown as yellow colored colonies, whereas cells with inactive FDHN remain acidic and form red colonies [16]. Our results (Fig. 2 right) are in agreement with the data from the benzyl viologen assays and confirm that FDH formation in the E. coli JS1 strain can be restored by the simultaneous presence of PSTK and SepCysS. This suggests that in E. coli SepCysS recognizes Sep-tRNASec as a substrate as do SepSecS and SelA [19].

3.2 SepCysS does not form Sec-tRNASec

The observation that the JS1 strain transformed with SepCysS and PSTK shows less FDHH activity compared to the SepSecS and PSTK complemented strain can be explained in two ways; (i) the SepCysS/PSTK complemented strain produces less selenoprotein, or (ii) SepCysS forms Cys-tRNASec, leading to cysteine incorporation at the Sec codon generating the sulfur homolog of FDHH. It is known that a Sec to Cys mutation in FDHH causes a 110-fold decrease in kcat compared to the wild type Sec-containing enzyme [20], and that Cys and Sec are the only amino acids that confer activity to FDHH.

To distinguish between these two possibilities, we carried out [75Se] in vivo labeling experiments. Transformed JS1 strains were grown anaerobically in the presence of formate and [75Se]-selenite. Total cell lysates, prepared from equal amounts of cells, were separated by SDS-PAGE. Radioactively labeled selenoprotein was detected by autoradiography. Our results (Fig. 3) show that [75Se] labeled proteins only occur in the ΔselA JS1 strain transformed either with E. coli selA or with both PSTK and SepSecS genes. Comparison of lanes 5 and 6 in Fig. 3 shows that under our experimental in vivo conditions SepCysS is unable to use the Se-donor selenophosphate as substrate. This is in contrast to SepSecS which accepts both thiophosphate and selenophosphate as substrates [4]. The currently available crystal structures of SepCysS [21] and SepSecS [4,10,22] do not offer any insight on this difference in substrate selection.

Fig. 3. Metabolic labeling of transformed ΔselA strains with 75Se.

Fig. 3

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The E. coli ΔselA strain JS1 was complemented with E. coli selA (lane 1), empty vector control (lane 2), M. jannaschii spcS (coding for SepSecS, lane 3), M. jannaschii pscS (coding for SepCysS, lane 4), M. jannaschii spcS and pstK genes (lane 5), and M. jannaschii pscS and pstK (lane 6). Two major bands were observed in the positive control lane 1. Based on the molecular weight marker, the upper band corresponds to FDHH. The lower band is likely a degradation product of FDHH, the sole selenoprotein in E. coli in the indicated growth conditions.

No [75Se] labeled proteins are detectable in transformants with PSTK and SepCysS genes, despite various efforts to increase the assay sensitivity (see Materials and Methods for the conditions tested). This suggests that explanation (ii) is correct, and thus SepCysS forms Cys-tRNASec in E. coli. To further confirm this result, we performed transformation assays in cells devoid of the selenium donor selenophosphate.

3.3 SepCysS forms Cys-tRNASec in E. coli

The E. coli selD gene encodes selenophosphate synthetase, the enzyme that forms selenophosphate using selenide and ATP. Selenophosphate is the activated selenium donor required for Sec-tRNASec synthesis [23]. Deletion of selD abolishes selenoprotein formation but does not affect sulfur metabolism since SelD is specific for selenide [24,25]. To further confirm that transformation of the ΔselA strain JS1 by the SepCysS and PSTK genes does not generate selenocysteine synthesized from selenophosphate, we constructed the E. coli ΔselA ΔselD double deletion strain MH1. This strain was then transformed with the SepCysS/PSTK or SepSecS/PSTK genes, and the resulting transformants were tested for FDHH activity with the benzyl viologen assay. Our results show that the SepSecS and PSTK genes are no longer able to restore FDHH activity in the ΔselA ΔselD double deletion strain MH1 (Fig. 4). However, the SepCysS and PSTK genes retain their ability to restore FDHH activity albeit somewhat weaker compared to the positive control (MH1 strain transformed with E. coli selA and selD). These results prove that the observed FDHH activity is due to a Cys active site residue and not due to a Sec one. Deletion of SelD in the MH1 strain precludes SepSecS from acting onto Sep-tRNASec since SelD is the only enzyme responsible for selenophosphate formation. On the other hand, SepCysS is still able to use an available sulfur donor and convert Sep-tRNASec to Cys-tRNASec. Although we do not show direct evidence for the presence of the sulfur homolog of FDHH, our findings are in agreement with previous reports showing that, other than Sec in the catalytic site of FDHH, only Cys can retain partial activity [20,26]. Taken together, these data imply that, in the presence of PSTK, SepCysS forms Cys-tRNASec in E. coli.

Fig. 4. SepCysS restores FDHH activity in an E. coli ΔselA ΔselD strain.

Fig. 4

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The indicated proteins (right) complement the loss of SelA and SelD in the E. coli strain MH1. Activity of the selenoprotein FDHH is tested with benzyl viologen assay.

4. Discussion

4.1 Aminoacyl-tRNA recognition by SepCysS

Our results indicate that SepCysS can convert Sep-tRNASec to Cys-tRNASec in E. coli. Its physiological function in methanogens is to convert Sep-tRNACys to Cys-tRNACys. Thus, SepCysS is the first enzyme involved in tRNA-dependent amino acid transformations shown to have the ability to act on two distinct tRNAs, tRNACys and tRNASec. Clearly, SepCysS must specifically recognize the phosphoserine moiety attached to either tRNACys or tRNASec. Indeed, SepCysS alone does not complement the E. coli ΔselA strain JS1 suggesting that Ser-tRNASec is not a substrate for SepCysS. This is not surprising given that precise recognition of the phosphate moiety of Sep is also the mode by which SepSecS discriminates against Ser-tRNASec [4]. The highly divergent sequences and structures of tRNACys and tRNASec imply that SepCysS does not rely heavily on tRNA identity for activity and strengthens the notion that Sep recognition is the main binding force of Sep-tRNA to the enzyme.

All known tRNACys species adopt the canonical 12 bp acceptor/TΨC helix, while tRNASec folds into the distinct 13 bp acceptor/ TΨC conformation that is crucial for recognition by SepSecS and most likely by all Sec-specific enzymes [4,27]. Thus, unlike its importance for SepSecS recognition, the unique structure of tRNASec does not appear to be an anti-determinant for SepCysS. We can speculate that apart from the phosphoserine moiety SepCysS is also recognizing the tip of the acceptor stem of E. coli tRNASec. In fact, though M. jannaschii tRNACys and E. coli tRNASec have different discriminator bases (U73 and G73, respectively), they do share a common first bp (G1-C72) at their acceptor stems. The benzyl viologen complementation assay has been proven very powerful in characterizing at least two of the enzymes (PSTK and SepSecS) involved in archaeal and eukaryal Sec biosynthesis [4,10,13]. In both cases, in vivo complementation results always correlated with in vitro results by purified enzymes, attesting to the capability of the benzyl viologen assay to render reliable data. Given the present lack of knowledge regarding the nature of the sulfur donor in the SepCysS reaction [9,11], the benzyl viologen assay may be useful for an in vivo study of the tRNACys and tRNASec recognition by SepCysS.

4.2 Aminoacyl-tRNA specificity of SelB

Cys-tRNASec formed by SepCysS in E. coli is incorporated during protein synthesis in response to the in frame UGA codon in the gene encoding formate dehydrogenase. Given the inability of EF-Tu to bind to tRNASec [6,28], we conclude that the specialized elongation factor SelB is transferring Cys-tRNASec to the translation apparatus. SelB is known to discriminate against unacylated tRNASec and Ser-tRNASec both in vivo [7] and in vitro [29,30]. SelB binds Sec-tRNASec one thousand times (Kd 0.2 pM) tighter than tRNASec or Ser-tRNASec (Kd 0.5 μM) in vitro [30] and it does not deliver Ser-tRNASec to the ribosome in vivo [7]. The higher affinity of SelB for Sec-tRNASec is attributed to the amino acid binding pocket of SelB that is specifically designed to bind Sec [7]. Our results suggest that Cys can be bound tightly enough in the active site of SelB to allow for Cys-tRNASec delivery to the ribosome. Thus, SelB adds to the list of enzymes that cannot entirely distinguish sulfur from selenium, such as SepSecS which uses thiophosphate in vitro to form Cys-tRNASec [4,10] and CysRS which can acylate Sec onto tRNACys [31,32].

4.3 Amino acid ambiguity for UGA decoding in methanogens

The physiological significance of Cys-tRNASec in organisms that possess both SepCysS and SepSecS is unclear at this point. In fact, Cys-tRNASec would only be formed if SepCysS successfully competes with SepSecS for binding to Sep-tRNASec. This would in turn mean that the UGA codon is ambiguous in methanogens as it would encode for both Cys and Sec during translation of the same open reading frame. In such a case, methanogenic selenoproteins would also be expressed with a Cys residue in place of Sec. This may be advantageous in cases of selenium deficiency in the environment since the cysteine homologs of most known selenoproteins are active albeit to a lesser extent than their selenium-containing counterparts [20,33,34]. Further investigation of the SepCysS activity towards Sep-tRNASec in Sec decoding archaea will shed light on the physiological importance of Cys-tRNASec and the in vivo role of the UGA codon in these organisms.

Acknowledgments

M.J.H. held a Feodor Lynen Postdoctoral Fellowship of the Alexander von Humboldt Stiftung, R.L.S. was supported by a Ruth L. Kirschstein National Research Service Award postdoctoral fellowship from the National Institute of General Medical Sciences, and S.P. holds a fellowship of the Yale University School of Medicine MD/PhD Program. This work was supported by grants from the Department of Energy, the National Institute for General Medical Sciences, and the National Science Foundation.

Abbreviations

aa-tRNA

aminoacyl-tRNA

EF-Tu

elongation factor Tu

FDHH

formate dehydrogenase H

FDHN

formate dehydrogenase N

IPTG

isopropyl-D-thiogalactoside

PLP

pyridoxal phosphate

PSTK

phosphoseryl-tRNASec kinase

Sec

selenocysteine

SelA

selenocysteine synthase

SelB

elongation factor SelB

SelD

selenophosphate synthetase

Sep

O-phosphoserine

SepCysS

Sep-tRNA:Cys-tRNA synthase

SepSecS

Sep-tRNA:Sec-tRNA synthase

Footnotes

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