Selenocysteine
A. Böck* and M. Thanbichler
[SECTION EDITOR: GEORGES N. COHEN]
Posted April 12, 2004
Department of Biology I, Microbiology, University of Munich, D-80638 Munich, Germany
Phone: 49-89-2180-6116, Fax: 49-89-2180-63857, E-mail:
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About 50 years ago, research on the biological function of the element selenium was initiated by the report of J. Pinsent (64) that generation of formate dehydrogenase activity by Escherichia coli requires the presence of both selenite and molybdate in the growth medium. Although this observation was rapidly followed by the discovery of a trace element function for selenium in mammals (72), it was nearly 20 years before it was demonstrated that this element is a constituent of selected bacterial and eukaryal enzymes (1 , 22, 67, 81) and also of defined tRNA species (84). Finally, in 1976, the chemical nature of the selenium constituent of a clostridial enzyme was disclosed as 2-seleno-alanine (trivial name, selenocysteine) (16), followed by the report that this nonstandard amino acid is also present in the formate dehydrogenase of Methanococcus vannielii (46). As for E. coli, Enoch and Lester proved that selenium is a component of the large subunit of formate dehydrogenase N (FDHN), which oxidizes formate and delivers the electrons to nitrate in the process of nitrate respiration (21). The rapid progress since then in our knowledge of the biochemistry and molecular biology of selenium in general, and specifically in E. coli, has rested on two seminal contributions. First, Cox et al. (18) devised a radiolabeling technique for the detection of selenium-containing macromolecules in E. coliwhich revealed that the organism synthesizes at least two selenopolypeptides, which were later identified as the large subunit of FDHN and as FDHH, which is a component of the formate hydrogen-lyase system. This finding was succeeded in 1991 by the discovery of FDHO, a third isoenzyme coupling formate oxidation to O2 reduction (71). The second important contribution was the development by several groups of rapid screening techniques for the isolation of mutants deficient in formate metabolism and, subsequently, the collection of a considerable number of such strains. Many of the isolates had pleiotropic lesions, preventing the formation of the activities of all three FDH isoenzymes (6, 14, 31, 32, 57), whereas several of them turned out to be blocked either in either selenocysteine biosynthesis or incorporation (51). Cloning and sequence analysis of the corresponding genes and of the structural genes for the three FDHs revealed that selenocysteine is cotranslationally incorporated in response to defined UGA codons. This observation was the first example of an expansion of the genetic code, which prompted the designation of selenocysteine as the 21st amino acid (11). The topic has been covered by a considerable number of reviews, among which several recent ones are relevant for the bacterial system (8, 10, 15, 35, 44, 48, 74).
In nature, selenium is predominantly associated with sulfur minerals, the Se/S ratios of which vary widely depending on the geological formation. Because of the chemical similarity between the two elements, selenium can intrude into the sulfur pathway at high Se/S ratios and can be statistically incorporated into polypeptides (Fig. 1) (17, 33, 39, 40, 41, 82). As shown in Fig. 1, free cysteine, as well as free selenocysteine, are synthesized via the cysteine biosynthetic pathway and thus enter the cysteine pool (61). The flow of the two analogs from this pool into protein either via methionine or by direct charging to tRNACys (85) is determined by the specificity and kinetic efficiency of cysteyl-tRNACys synthetase and the enzymes leading the flux in the direction of cystathionine. The ratio between selenomethionine and selenocysteine randomly incorporated into protein may therefore vary depending on the organism (27, 32, 58). In this way, AUG is decoded with methionine or selenomethionine and UGU and UGC are decoded with cysteine or selenocysteine. Whereas the replacement of methionine by selenomethionine is tolerated by the organism, substitution of selenocysteine for cysteine is harmful (17, 40, 41). Nevertheless, systems for replacement of methionine by selenomethionine or cysteine by selenocysteine have been developed and are important tools to facilitate the structural analysis of proteins by nuclear magnetic resonance spectroscopy (62, 63) or X-ray crystallography (38).
Considerable efforts have been made to replace a single cysteine with a selenocysteine or vice versa. The introduction of a selenocysteine in place of a cysteine by random incorporation is technically feasible if only one such residue is present in the polypeptide. Statistical exchange of the catalytic cysteine of the glyceraldehyde dehydrogenase from Bacillus stearothermophilus,for example,resulted in an enzyme with peroxidase activity (12). In contrast, targeted insertion of selenocysteine at the ribosome could be achieved only when a UGA was introduced in place of a cysteine codon and the downstream nucleotide sequence could be concomitantly modified in a such a way that it mimicked the structure of the cognate selenocysteine insertion sequence (SECIS) element (34) or when the SECIS element could be positioned in the 3' untranslated region (2).
The replacement of a selenocysteine by a cysteine residue, on the other hand, requires only the substitution of a cysteine codon for the UGA. Several sulfur homologs of selenoenzymes have been created in this way. A sulfur form of FDHH has been generated and purified, and its activity was compared with that of the purified Se form. It was found that the substrate affinity was only slightly affected but that there was a dramatic decrease in the reaction rate upon replacement of the Se by S, possibly due to the lower chemical reactivity of the thiol at physiological pH (3).
At low Se/S ratios and concentrations well below the micromolar range, selenium is incorporated specifically into the so-called selenoproteins or into a modified base (5-methylaminomethyl-2-seleno-uridine) of selected tRNAs. These processes require the synthesis of an activated selenium species, monoselenophosphate, catalyzed by selenophosphate synthetase, the product of the selD (19, 30, 53, 83) (previously fdhB) gene (32). Selenophosphate serves as a selenium donor for both the conversion of 5-methylaminomethyl-2-thio-uridine into 5-methylaminomethyl-2-seleno-uridine (83) and that of seryl-tRNASec into selenocysteyl-tRNASec (53), which is used for decoding the specialized UGA codons of selenoprotein mRNAs with selenocysteine (Fig. 1, specific reactions). The breakdown of selenocysteine is reviewed in another EcoSal module.
The central macromolecule for the synthesis and incorporation of selenocysteine (see Fig. 3 for a summary) is a specialized tRNA, designated tRNASec. It is the product of the selC (51, 52) (previously fdhC) (31) gene. tRNASec fulfils a multitude of functions, which are based on its unique structural properties, compared to canonical elongator RNAs (15).
Canonical elongator tRNA species possess 22 invariant or semi-invariant nucleotides; their secondary structure is characterized by a 7-bp-long aminoacyl acceptor stem, a 3- or 4-bp-long D stem, and a 5-bp-long anticodon stem, as well as a 5-bp-long T stem. The D loop comprises between 8 and 10 nucleotides, whereas the anticodon and the T loops consist of 5 nucleotides each. The length of the variable region distinguishes two classes of elongator tRNAs: class I species have a short variable region made up of 4 or 5 nucleotides, whereas class II molecules have a variable region of 10 to 24 nucleotides (15).
Figure 2gives a schematic representation of the structure of tRNASec from E. coli and highlights the features that deviate from the invariant properties of canonical elongator tRNA species (5). Although tRNASec clearly belongs to class II of tRNA molecules, several of the conserved positions are no longer maintained, most prominantly U8 and A14. Even more striking differences exist in the secondary structure: first, the D stem is extended to a 6-bp helix, which restricts the D loop to only 4 nucleotides. The acceptor stem is extended by 1 bp, resulting in a 13-bp acceptor arm. Because of the elongated D stem, the number of long-range tertiary interactions is restricted to three, resulting in a loosened interaction between the acceptor helix and the anticodon helix in the three-dimensional structure. Finally, because of the particularily long extra arm and the extended acceptor stem, tRNASec is the largest tRNA in E. coli, and also in other organisms (15, 44, 52).
tRNASec possesses the discriminator base G73 and the identity elements of serine-specific tRNA isoacceptors(Fig. 2). Consequently, it is charged with serine by seryl-tRNA synthetase, which also aminoacylates serine isoacceptors. The overall charging efficiency, however, is only 1% of that of cognate serine-specific tRNAs, which results from the reduced ligand affinity and kcat of the synthetase. The structural features responsible for the decreased catalytic efficiency are unknown, but the decrease may reflect the low product requirement, since there are only three differentially synthesized selenoproteins in the E. coli cell, each of which has only one selenocysteine residue.
The conversion of seryl-tRNASec into selenocysteyl-tRNASec is catalyzed by selenocysteine synthase (29) (Fig. 3), the product of the selA gene (previously the fdhA locus [32], which was later shown to harbor two genes, selA and selB) (52). Selenocysteine synthase is a homodecameric enzyme made up of two pentameric rings stacked on top of each other (20). Each monomer possesses a pyridoxal-5'-phosphate prosthetic group, which is linked to lysine-295. Two 50-kDa subunits (one from each ring) bind one molecule of seryl-tRNASec, so that the fully loaded enzyme is complexed with five charged tRNA molecules (20, 24). The exchange of the hydroxyl against a selenol group by selenocysteine synthase (SS) formally takes place in a two-step reaction: (i) seryl-tRNASec + SS → dehydroalanyl-tRNASec-SS + H2O and (ii) dehydroalanyl-tRNASec + HSe-PO3 2−→ selenocysteyl-tRNASec + PO4 3−+ SS.
In the first step, the 2-amino group of the seryl moiety forms a Schiff base with the carbonyl of the pyridoxal-5'-phosphate cofactor, resulting in the 2,3-elimination of a water molecule and the formation of a dehydroalanyl intermediate. In the second step, nucleophilic addition of HSe− from selenophosphate as a donor yields selenocysteyl-tRNASec (35). The interaction of selenocysteine synthase with its tRNA substrate must involve the body of the tRNA molecule, as seryl-tRNASer is not accepted as a substrate. This also means that the identity elements for serylation by seryl-tRNA synthetase and for recognition by selenocysteine synthase comprise separate structural features of the tRNA. Free selenide, and also sulfide, can serve as nucleophiles, albeit with very low affinity and catalytic efficiency (80).
The tRNA-coupled biosynthesis of selenocysteine is reminiscent of the formylation of methionyl-tRNA by methionyl-tRNA transformylase and the formation of glutaminyl- and asparaginyl-tRNA from glutamyl- and aspartyl-tRNA by the respective amidotransferases (45).
The incorporation of selenocysteine follows a path different and separate from that involved in classical protein synthesis (Fig. 3). It is based on the redefinition of a stop codon—UGA—as the sense codon for selenocysteine and the complex activity of a specialized translation elongation factor—SelB—taking over the function of the canonical factor EF-Tu.
Selenocysteine insertion is determined by the stop codon, UGA, in selenoprotein mRNAs from organisms belonging to all three domains of life (13, 86). This violates the basic rule that within one cell no codon can have more than one meaning. Consequently, additional features of the mRNA must determine that this particular UGA codon is to be read as sense and that it does not serve as a signal for chain termination. The choice of E. coli as a working system was fortunate, since in contrast to the situation seen in other organisms, like gram-positive bacteria (28, 29), this feature could be clearly attributed to an ~40-nucleotide-long conserved hairpin following the UGA codon on the 3' side (36, 87, 88) (Fig. 4C). The designation SECIS element was adopted for it following the suggestion of a homologous structure in eukaryal selenoprotein mRNAs (59). Extensive genetic analysis showed that the apical part of the stem-loop structure, consisting of 17 nucleotides (the so-called "minihelix"), is sufficient for its function, although the lower part of the helix is required for full efficiency (36, 53). Point mutations and deletions reaching into the SECIS element from the 3' side abolished SECIS function when the minihelix was affected. Similarly, increasing or decreasing the optimal distance of 11 bp between the UGA codon and the minihelix drastically reduced the efficiency of selenocysteine incorporation. Clearly, therefore, the context of the mRNA at the 3' side is the determinant that causes the UGA to be read as sense.
Selenocysteyl-tRNASec does not interact with EF-Tu with significant affinity (23). The lack of recognition has been correlated with an antideterminant region present in the acceptor arm of the tRNA and consisting of the last base pair of the aminacyl acceptor stem and the first 2 bp of the T stem (Fig. 2). This motif is conserved in all tRNASec species and does not occur in ordinary elongator tRNAs (68). Instead, the role of EF-Tu is taken over by a specialized elongation factor, designated SelB, which is the product of the promoter-distal gene of the selAB operon (25). The N-terminal two-thirds of SelB displays distinct sequence similarity to domains I, II, and III of EF-Tu (Fig. 4A). The homology of the two proteins is further supported by biochemical results showing that SelB has functions similar to those of EF-Tu: it interacts both with guanosine nucleotides and with selenocysteyl-tRNASec (4, 25, 36, 44, 49). Seryl-tRNASec, the biosynthetic precursor, does not interact with the SelB protein, and this appears to be the primary basis for discrimination between the 2 amino acids during selenocysteine incorporation. The mechanism by which the protein differentiates between the seryl and the selenocysteyl moieties of the tRNA is still unresolved.
The 25-kDa C-terminal extension of SelB, designated domain IV, is essential for its function in selenoprotein synthesis, since truncated SelB molecules containing only domains I, II, and III are inactive (79). It was found that domain IV binds to the complete SECIS element, as well as to its minihelical part; the binding domain could be localized to the ultimate 17-kDa part, designated domain IVb (Fig. 4A) (49). The specificity of the interaction was investigated by analysis of the effects of mutations in the SECIS motif (28, 36, 55), by probing the protection by SelB of SECIS RNA against chemical modification and enzymatic cleavage (4, 44, 66), by analysis of the suppression of SECIS mutations by compensatory mutations in selB (50), and by selection of aptamers from a randomly synthesized RNA pool binding to SelB (47). The results show that SelB contacts the SECIS element via the bulged-out U of the stem and a GU duplet in the apical loop, which are highly exposed, as shown by structural analyses (27, 42). The investigation of SelB mutants compensating for the defects of mutant SECIS elements identified a 20-amino-acid stretch of the protein within domain IVb that might be part of the contact site between the protein and the RNA. Finally, about half of the aptamers selected from a random pool in the SELEX approach possessed the wild-type sequence. Intriguingly, there were representatives among the other half that had a higher affinity for the protein than the genuine element, but all of them lacked biological activity. This means that binding to SelB and promotion of UGA readthrough are not necessarily coupled processes. Rather, some consequence of the correct interaction appears to be an essential requirement. On the other hand, systematic screening of a randomized SECIS library for elements that are able to mediate selenocysteine insertion revealed that even structures that significantly deviate from the consensus can be functional in vivo (70).
The crucial event in the decoding process consists of the formation of a quaternary complex of SelB with GTP, selenocysteyl-tRNASec, and the SECIS element (4, 36). Binding of the two RNA ligands is strongly cooperative, as detected by electromobility shift experiments (4) and stopped-flow measurements involving purified components. Rapid kinetic analysis of the SelB-SECIS interaction revealed that the presence of the selenocysteylated tRNASec increases the affinity of the protein for the SECIS element nearly 10-fold by reduction of the rate of dissociation (78). A recombinant protein comprising subdomains IVa and IVb displays the same interaction characteristics with the SECIS minihelix as the entire SelB molecule in the absence of the tRNA. In contrast, recombinant subdomain IVb alone possesses an affinity for the SECIS element identical to that of the entire protein complexed with selenocysteyl-tRNASec (78). This forces the conclusion that within the quaternary complex binding of selenocysteyl-tRNASec confers a conformation on SelB that results in an increased affinity to the mRNA, presumably by interrupting inhibitory interactions that subdomain IVa exerts on subdomain IVb.
It appears that the conformation attained is required for productive interaction with the ribosomal A site according to the following scenario. SelB bound to a selenoprotein mRNA and carrying GTP and selenocysteyl-tRNASec is approached by a translating ribosome. The contact with the ribosome, most probably via productive codon-anticodon interaction, induces GTP hydrolysis by SelB and the subsequent release of the tRNA in the vicinity of the A site. Loss of the tRNA then decreases the affinity of SelB for the SECIS element, and the protein leaves the mRNA to free it for the translation of downstream codons. The processes involved in decoding a single UGA codon as selenocysteine stall the ribosome for ~8 s, a time that usually allows the polymerization of ~100 standard amino acids (76).
In summary, binding of SelB to the mRNA at the SECIS element in bacteria not only tethers the rare tRNA to the site of its codon but also keeps SelB in a conformation that is able to interact with the ribosome. This mechanism ensures that ordinary stop codons are not translated with selenocysteine, because SelB cannot donate the tRNA to the ribosome in the absence of the cognate mRNA structure. Experimental evidence for this decoding procedure has been provided by measuring GTP hydrolysis by SelB in the presence of purified ribosomes (43). The intrinsic GTPase activity of SelB was only marginally stimulated by ribosomes in the absence of mRNA. Addition of synthetic selenoprotein mRNA stimulated the activity greatly, and the same stimulation was observed when only the 17-nucleotide-long minihelix, i. e., the minimal ligand, was present.
Insight into the nature of the conformational change induced by binding of the RNA ligands has been obtained recently by solution of the X-ray structure of domain IV of SelB from Moorella thermoacetica (73). Although subdomains IVa and IVb do not show significant sequence similarity, they display a striking similarity in the overall three-dimensional structure. The two subdomains are connected by a flexible hinge region and interact with each other, probably via ionic interactions, which results in an L-shaped arrangement.
Whereas the biochemical basis for the redefinition of a stop codon as a sense codon appears to be satisfactorily explained by the properties and functions of the SelB quaternary complex, the reason why translation termination is efficiently suppressed at the particular UGA codons is less well understood. Early experiments showed that translation of the UGA competes with termination, since overexpression of fdhF, the gene for the selenoprotein of FDHH, yielded a truncated gene product of the size expected if the UGA was used as a stop codon. This truncated variant was not produced when UGA was converted into an ordinary sense codon (52). Even more compelling evidence for such competition came from expression of the gene for mammalian thioredoxine reductase (TR) in E. coli. In TR, selenocysteine is the penultimate amino acid at the C terminus. Overexpression of the TR gene harboring the E. coli fdhF SECIS element in its 3' untranslated region resulted in a gene product that lacked the last 2 amino acids, which indicates that UGA functioned as a stop signal. However, when the level of the selenoprotein synthesis machinery was raised concomitantly, the ratio of prematurely terminated product was significantly reduced in favor of the full-size protein containing selenocysteine (2). Readthrough over the UGA thus requires balanced amounts of the mRNA and the components of the selenocysteine insertion machinery.
The present view of the polypeptide chain termination process is not restricted to the function of the termination codon and its interaction with the release factors. Other determinants involved are the nature of the next 4 bases following the stop codon on the 3' side and the chemical nature of the 2 amino acids encoded immediately upstream of the stop codon. Of particular importance, a C or an A in the fourth position, as predominantly found in selenoprotein mRNAs, reduces termination efficiency (65), whereas a conserved C immediately following the UGA promotes termination in the absence of selenium (56, 69). The 2 amino acids preceding selenocysteine in the nascent peptide can influence the efficiency of termination, depending on their hydrophobicities (9, 56, 60). It has been shown that termination at the UGA selenocysteine codon follows the same rules as ordinary stop codon recognition. Thus, both the concentrations of the components involved and the sequence context determine whether the UGA of a selenoprotein mRNA is read as a stop codon or as a sense codon (58). Indirectly, this also implies a dependence on physiological parameters like the growth rate, since the compositions of bacterial cells may vary greatly in different metabolic states.
The three FDHs mentioned above are the only selenoproteins synthesized by E. coli(7, 71, 75, 86). FDHO is formed under both aerobic and anaerobic respiratory conditions, FDHN is present in nitrate-reducing cells only, and FDHH is a constituent of the formate hydrogen-lyase system, which is expressed solely during fermentation. Although the synthesis of selenoproteins is regulated according to physiological needs, it was found that the transcription of the genes coding for the selenocysteine insertion machinery is constitutive (71). This also holds for the translation of the selD mRNA, whose product synthesizes selenophosphate, which is an essential substrate for both selenocysteine biosynthesis and tRNA modification. On the other hand, translation of the selAB mRNA (Fig. 4B) coding for selenocysteine synthase and the SelB protein is regulated by repression involving the SelB protein and selenocysteyl-tRNASec (77).
The crucial element for the regulation is a putative secondary structure at the 5' end of the untranslated region of the selAB mRNA. It resembles the SECIS element of selenoprotein mRNAs and has been designated SECIS-like (Fig. 4C). The SECIS-like element binds translation factor SelB and also undergoes quaternary complex formation with selenocysteyl-tRNASec. Introduction of mutations in the loop region of the SECIS-like motif that are detrimental to the function of genuine SECIS elements abolishes binding. When tested invivo, all conditions under which quaternary complex formation is prevented or reduced lead to increased formation of the selAB gene products. The generation and analysis of transcriptional and translational reporter gene fusions of selA and selB yield an expression pattern identical to that obtained by measuring the actual amounts of SelA and SelB proteins. Compared to SelA, SelB is less tightly controlled because of an internal ribosomal entry site upstream of the selB reading frame. This is physiologically significant, as a basal level of the regulatory molecule has to be maintained under all growth conditions.
The scheme depicted in Fig. 5 represents a novel type of control mechanism that senses the charging level of tRNASec. In this way, the availability of the trace element selenium is elegantly tied to the formation of the selenocysteine biosynthetic enzyme and translation factor SelB. There is no proven rationale for the need to control the expression of the selAB genes; one possible explanation, however, is that for quaternary complex formation the components need to be present in a balanced ratio. Indeed, overexpression of selB in an otherwise wild-type background completely blocks selenoprotein synthesis, and the blockade can be reversed by concomitantly overexpressing tRNASec (79).
The original work performed in our laboratory was supported by grants from the Deutsche Forschungsgemeinschaft and by the Bundesministerium für Forschung und Technologie via the Munich Gene Center.
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