Selenoprotein synthesis: an expansion of the genetic code.

A number of enzymes employ the unusual amino acid selenocysteine as part of their active site because of its high chemical reactivity. Selenocysteine is incorporated into these proteins co-translationally: biosynthesis occurs on a specific tRNA and insertion into a growing polypeptide is directed by a UGA codon in the mRNA. In E. coli, this requires a specific translation factor. Selenocysteine thus represents a unique expansion of the genetic code.     

Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SelB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SelB-C). SelB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SelB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SelB-RNA interaction is sequence independent, possibly reflecting SelB-tRNA/-rRNA recognitions. Based on these data, the dynamic SelB-ribosome-mRNA-tRNA interactions will be discussed.     

Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality

Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins. To assess a biological role for these proteins, a simple chemically defined medium that supports growth of adult Drosophila and requires selenium supplementation for optimal survival was devised. Flies survived on this medium supplemented with 10(-8) to 10(-6) m selenium or on the commonly used yeast-based complete medium at about twice the rate as those on a medium without selenium or with >10(-6) m selenium. This effect correlated with changes in selenoprotein mRNA expression. The number of eggs laid by Drosophila was reduced approximately in half in the chemically defined medium compared with the same medium supplemented with selenium. The data provide evidence that dietary selenium deficiency shortens, while supplementation of the diet with selenium normalizes the Drosophila life span by a process that may involve the newly identified selenoproteins.     

Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif

Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3'-untranslated region of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence data bases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities, and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed by in vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with mitochondrial thioredoxin reductase-2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pilus formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.     

硒蛋白的分子生物学研究进展

已有35种硒蛋白被分离和表征,但许多硒蛋白及其功能仍未完全阐明.硒半胱氨酸(Sec)作为参入蛋白质的第21种氨基酸,由硒蛋白mRNA上的UGA编码.在原核生物,Sec参入硒蛋白的复杂机制已经较为明确,需要四种基因产物（SELA、SELB、SELC和SELD）和一个存在于硒蛋白mRNA上的被称为Sec插入序列(SECIS)的茎环(stem loop)样二级结构.在真核生物,硒蛋白生物合成途径可能在SECIS的结构和位置、特异的延伸因子及其他RNA-RNA或RNA-蛋白质因子之间的相互作用等方面与原核生物不同.另外,哺乳动物硒蛋白mRNA上的UGA翻译为Sec的过程低效,特定位点的UGA密码子不同功能(终止密码和Sec密码)的调控可能是硒蛋白表达低效的关键.     

Targeted insertion of selenocysteine into the alpha subunit of formate dehydrogenase from Methanobacterium formicicum.

Selenocysteine incorporation into proteins is directed by an opal (UGA) codon and requires the existence of a stem-loop structure in the mRNA flanking the UGA at its 3' side. To analyze the sequence and secondary-structure requirements for UGA decoding, we have introduced mutations into the fdhA gene from Methanobacterium formicicum, which codes for the alpha subunit of the F420-reducing formate dehydrogenase. The M. formicicum enzyme contains a cysteine residue at the position where the Escherichia coli formate dehydrogenase H carries a selenocysteine moiety. The codon (UGC) for this cysteine residue was changed into a UGA codon, and mutations were successively introduced at the 5' and 3' sides to generate a stable secondary structure of the mRNA and to approximate the sequence of the predicted E. coli fdhF mRNA hairpin structure. It was found that introduction of the UGA and generation of a stable putative stem-loop structure were not sufficient for decoding with selenocysteine. Efficient selenocysteine incorporation, however, was obtained when the loop and the immediately adjacent portion of the putative stem had a sequence identical to that present in the E. coli fdhF mRNA structure.     

Structural basis for mRNA recognition by elongation factor SelB

In bacteria, incorporation of selenocysteine, the 21(st) amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 A. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.     

The bulged nucleotide in the Escherichia coli minimal selenocysteine insertion sequence participates in interaction with SelB: a genetic approach

The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a cis-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In Escherichia coli, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the fdhF SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the E. coli fdhF SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing selB mutations that suppress mutations of the bulged nucleotide in the SECIS. All the selB suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These selB suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the E. coli SECIS. Thus, the E. coli SECIS upper stem-loop structure can be considered a "single suppressible unit," suggesting that there is some flexibility to the nature of the interaction between this element and SelB.     

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.     

cDNA cloning,expression pattern and RNA binding analysis of human selenocysteine insertion sequence (SECIS) binding protein 2

Selenocysteine and selenoprotein synthesis require a complex molecular machinery in mammals. Among the key players is the RNA-protein complex formed by the selenocysteine insertion sequence (SECIS) binding protein (SBP2) and the SECIS element, an RNA hairpin in the 3' untranslated regions of selenoprotein messenger RNAs (mRNAs). We have isolated the DNA complementary to mRNA of the human SBP2, enabling us to establish that it differs from a previously reported human SBP2-like protein. Examination of the expression pattern revealed that the human SBP2 protein is encoded by a 4 kb long mRNA that is over-expressed in testis. Compared to the rat SBP2 sequence, the human SBP2 protein displays two highly conserved domains with 92 and 95% amino acid identity, the latter one containing the RNA binding domain. The inter-domain section carries 55% sequence identity, the remainder of the SBP2 sequences showing about 65% identity, values lower than expected for two mammalian proteins. Interestingly, we could show that the binding of human SBP2 to the SECIS RNA is stimulated by the selenoprotein-specialized elongation translation factor mSelB/eEFsec.     

A recoding element that stimulates decoding of UGA codons by Sec tRNA[Ser]Sec

Selenocysteine insertion during decoding of eukaryotic selenoprotein mRNA requires several trans-acting factors and a cis-acting selenocysteine insertion sequence (SECIS) usually located in the 3' UTR. A second cis-acting selenocysteine codon redefinition element (SRE) has recently been described that resides near the UGA-Sec codon of selenoprotein N (SEPN1). Similar phylogenetically conserved elements can be predicted in a subset of eukaryotic selenoprotein mRNAs. Previous experimental analysis of the SEPN1 SRE revealed it to have a stimulatory effect on readthrough of the UGA-Sec codon, which was not dependent upon the presence of a SECIS element in the 3' UTR; although, as expected, readthrough efficiency was further elevated by inclusion of a SECIS. In order to examine the nature of the redefinition event stimulated by the SEPN1 SRE, we have modified an experimentally tractable in vitro translation system that recapitulates efficient selenocysteine insertion. The results presented here illustrate that the SRE element has a stimulatory effect on decoding of the UGA-Sec codon by both the methylated and unmethylated isoforms of Sec tRNA([Ser]Sec), and confirm that efficient selenocysteine insertion is dependent on the presence of a 3'-UTR SECIS. The variation in recoding elements predicted near UGA-Sec codons implies that these elements may play a differential role in determining the amount of selenoprotein produced by acting as controllers of UGA decoding efficiency.     

Pyrrolysine and selenocysteine use dissimilar decoding strategies

Selenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies.     

Processive Selenocysteine Incorporation during Synthesis of Eukaryotic Selenoproteins

Selenoproteins are a family of proteins that share the common feature of containing selenocysteine, the “twenty-first” amino acid. Selenocysteine incorporation occurs during translation of selenoprotein messages by redefinition of UGA codons, which normally specify termination of translation. Studies of the eukaryotic selenocysteine incorporation mechanism suggest that selenocysteine insertion is inefficient compared with termination. Nevertheless, selenoprotein P and several other selenoproteins are known to contain multiple selenocysteines. The production of full-length (FL) protein from these messages would seem to demand highly efficient selenocysteine incorporation due to the compounding effect of termination at each UGA codon. We present data demonstrating that efficient incorporation of multiple selenocysteines can be reconstituted in rabbit reticulocyte lysate translation reactions. Selenocysteine incorporation at the first UGA codon is inefficient but increases by approximately 10-fold at subsequent downstream UGA codons. We found that ribosomes in the “processive” phase of selenocysteine incorporation (i.e., after decoding the first UGA codon as selenocysteine) are fully competent to terminate translation at UAG and UAA codons, that ribosomes become less efficient at selenocysteine incorporation as the distance between UGA codons is increased, and that efficient selenocysteine incorporation is not dependent on cis-acting elements unique to selenoprotein P. Furthermore, we found that the percentage of ribosomes decoding a UGA codon as selenocysteine rather than termination can be increased by 3- to 5-fold by placing the murine leukemia virus UAG read-through element upstream of the first UGA codon or by providing a competing messenger RNA in trans. The mechanisms of selenocysteine incorporation and selenoprotein synthesis are discussed in light of these results.     

Efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck

Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3' untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a "bottleneck," slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.     

Crystal structure of an mRNA-binding fragment of Moorella thermoacetica elongation factor SelB

SelB is an elongation factor needed for the co-translational incorporation of selenocysteine. Selenocysteine is coded by a UGA stop codon in combination with a specific downstream mRNA hairpin. In bacteria, the C-terminal part of SelB recognizes this hairpin, while the N-terminal part binds GTP and tRNA in analogy with elongation factor Tu (EF-Tu). We present the crystal structure of a C-terminal fragment of SelB (SelB-C) from Moorella thermoacetica at 2.12 A resolution, solved by a combination of selenium and yttrium multiwavelength anomalous dispersion. This 264 amino acid fragment contains the entire C-terminal extension beginning after the EF-Tu-homologous domains. SelB-C consists of four similar winged-helix domains arranged into the shape of an L. This is the first example of winged-helix domains involved in RNA binding. The location of conserved basic amino acids, together with data from the literature, define the position of the mRNA-binding site. Steric requirements indicate that a conformational change may occur upon ribosome interaction. Structural observations and data in the literature suggest that this change happens upon mRNA binding.     

The nature of the minimal 'selenocysteine insertion sequence' (SECIS) in Escherichia coli.

The UGA codon, usually a stop codon, can also direct the incorporation into a protein of the modified amino acid selenocysteine. This UGA decoding process requires a cis -acting mRNA element called 'selenocysteine insertion sequence' (SECIS) that can form a stem-loop structure. In Escherichia coli the SECIS of the selenoprotein formate dehydrogenase (FdhH) mRNA has been previously described to consist of at least 40 nucleotides following the UGA codon. Here we determined the nature of the minimal SECIS required for the in vivo UGA-directed selenocysteine incorporation in E.coli . Our study is based on extensive mutational analysis of the fdhF SECIS DNA located in a lac' Z fusion. We found that the whole stem-loop RNA structure of the E.coli fdhF SECIS previously described is not required for the UGA-directed selenocysteine incorporation in vivo . Rather, only its upper stem-loop structure of 17 nucleotides is necessary on the condition that it is located in a proper distance (11 nucleotides) from the UGA codon. Based on these observations, we present a new model for the minimal E.coli SECIS.     

An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure

The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading. Interesting alternative relates to the function of the UGA codon. Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide. UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure. Here we discuss our investigation on the E. coli SECIS. This includes the follows: 1) The nature of the minimal E. coli SECIS. We found that in E. coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA [34]; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation [34] because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency [35]. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Finally, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.     

硒蛋白合成的特殊机制

硒蛋白含有一种特殊氨基酸--硒代半胱氨酸。在翻译阶段,该氨基酸从硒蛋白ｍＲＮＡ编码区的ＵＧＡ密码子处掺入多肽链。已证明它由丝氨酸和活性硒供体分子合成。一种独特的ｔＲＮＡ、某些特殊蛋白质因子以及硒蛋白ｍＲＮＡ的特殊二级结构是ＵＧＡ解读为硒代半胱氨酸所必需的。     

Molecular biology of selenium with implications for its metabolism.

Selenium has a highly specific metabolism centered around its incorporation as selenocysteine into selenoproteins. An outline of this metabolism has emerged from recent molecular biological and biochemical studies of bacteria and animals. A unique tRNA, designated tRNA[Ser]Sec, is charged with L-serine, which is then converted through at least two steps to selenocysteine. With the aid of a unique translation factor, the selenocysteinyl-tRNA[Ser]Sec recognizes specific UGA codons in mRNA to insert selenocysteine into the primary structure of selenoproteins. Turnover of selenoproteins presumably liberates selenocysteine which is toxic in its free form. Selenocysteine beta-lyase catabolizes free selenocysteine and makes its selenium available for reuse. Proteins contain almost all the selenium in animals. Of the known selenoproteins, the glutathione peroxidases contain the most selenium. Cellular and plasma glutathione peroxidases are products of different genes but have 44% identity of amino acid sequence. There is evidence for other proteins of this family. Selenoprotein P is an unrelated protein with multiple selenocysteines in its primary structure. It contains most of the selenium in rat plasma. Studies of the regulation of cellular glutathione peroxidase by selenium have yielded conflicting results, but there is a strong suggestion that mRNA levels of the rodent liver glutathione peroxidase decrease in selenium deficiency. This could be a mechanism for directing selenium to the synthesis of other selenoproteins. Although present knowledge allows construction of an outline of selenium metabolism, several steps have not been characterized and little is known about mechanisms of its regulation.     

SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.