Titration and Conditional Knockdown of the prfB Gene in Escherichia coli: Effects on Growth and Overproduction of the Recombinant Mammalian Selenoprotein Thioredoxin Reductase

Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P_BAD promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P_BAD-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.     

Wobble decoding by the Escherichia coli selenocysteine insertion machinery

Selenoprotein expression in Escherichia coli redefines specific single UGA codons from translational termination to selenocysteine (Sec) insertion. This process requires the presence of a Sec Insertion Sequence (SECIS) in the mRNA, which forms a secondary structure that binds a unique Sec-specific elongation factor that catalyzes Sec insertion at the predefined UGA instead of release factor 2-mediated termination. During overproduction of recombinant selenoproteins, this process nonetheless typically results in expression of UGA-truncated products together with the production of recombinant selenoproteins. Here, we found that premature termination can be fully avoided through a SECIS-dependent Sec-mediated suppression of UGG, thereby yielding either tryptophan or Sec insertion without detectable premature truncation. The yield of recombinant selenoprotein produced with this method approached that obtained with a classical UGA codon for Sec insertion. Sec-mediated suppression of UGG thus provides a novel method for selenoprotein production, as here demonstrated with rat thioredoxin reductase. The results also reveal that the E. coli selenoprotein synthesis machinery has the inherent capability to promote wobble decoding.     

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA^[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.     

Eukaryotic selenoprotein synthesis: mechanistic insight incorporating new factors and new functions for old factors

Selenium is an essential micronutrient that has been linked to various aspects of human health. Selenium exerts its biological activity through the incorporation of the amino acid, selenocysteine (Sec), into a unique class of proteins termed selenoproteins. Sec incorporation occurs cotranslationally at UGA codons in archaea, prokaryotes, and eukaryotes. UGA codons specify Sec coding rather than termination by the presence of specific secondary structures in mRNAs termed selenocysteine insertion (SECIS) elements, and trans-acting factors that associate with SECIS elements. Herein, we discuss the various proteins known to function in eukaryotic selenoprotein biosynthesis, including several players whose roles have only been elucidated very recently.     

Eukaryotic selenocysteine incorporation follows a nonprocessive mechanism that competes with translational termination

The synthesis of eukaryotic selenoproteins involves the recoding of an internal UGA codon as a site for selenocysteine incorporation. This recoding event is directed by a selenocysteine insertion sequence in the 3'-untranslated region. Because UGA also functions as a signal for peptidyl-tRNA hydrolysis, we have investigated how the rates of translational termination and selenocysteine incorporation relate to cis-acting elements in the mRNA as well as to trans-acting factors in the cytoplasm. We used cis-elements from the phospholipid glutathione peroxidase gene as the basis for this work because of its relatively high efficiency of selenocysteine incorporation. The last two codons preceding the UGA were found to exert a far greater influence on selenocysteine incorporation than nucleotides downstream of it. The efficiency of selenocysteine incorporation was generally much less than 100% but could be partially enhanced by concomitant overexpression of the tRNA(Sec) gene. The combination of two or three UGA codons in one reading frame led to a dramatic reduction in the yield of full-length protein. It is therefore unlikely that multiple incorporations of selenocysteine are processive with respect to the mode of action of the ribosomal complex binding to the UGA site. These observations are discussed in terms of the mechanism of selenoprotein synthesis and its ability to compete with termination at UGA codons.     

Partitioning between recoding and termination at a stop codon–selenocysteine insertion sequence

Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNA^Sec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons.     

The Efficiency of Selenocysteine Incorporation Is Regulated by Translation Initiation Factors

Selenocysteine (Sec) incorporation is an essential process required for the production of at least 25 human selenoproteins. This unique amino acid is co-translationally incorporated at specific UGA codons that normally serve as termination signals. Recoding from stop to Sec involves a cis-acting Sec insertion sequence element in the 3′ untranslated region of selenoprotein mRNAs as well as Sec insertion sequence binding protein 2, Sec-tRNA^Sec, and the Sec-specific elongation factor, eEFSec. The interplay between recoding and termination at Sec codons has served as a focal point in researching the mechanism of Sec insertion, but the role of translation initiation has not been addressed. In this report, we show that the cricket paralysis virus intergenic internal ribosome entry site is able to support Sec incorporation, thus providing evidence that the canonical functions of translation initiation factors are not required. Additionally, we show that neither a 5′ cap nor a 3′ poly(A) tail enhances Sec incorporation. Interestingly, however, the presence of the internal ribosome entry site significantly decreases Sec incorporation efficiency, suggesting a role for translation initiation in regulating the efficiency of UGA recoding.     

A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.     

The prokaryotic selenoproteome

In the genetic code, the UGA codon has a dual function as it encodes selenocysteine (Sec) and serves as a stop signal. However, only the translation terminator function is used in gene annotation programs, resulting in misannotation of selenoprotein genes. Here, we applied two independent bioinformatics approaches to characterize a selenoprotein set in prokaryotic genomes. One method searched for selenoprotein genes by identifying RNA stem-loop structures, selenocysteine insertion sequence elements; the second approach identified Sec/Cys pairs in homologous sequences. These analyses identified all or almost all selenoproteins in completely sequenced bacterial and archaeal genomes and provided a view on the distribution and composition of prokaryotic selenoproteomes. In addition, lineage-specific and core selenoproteins were detected, which provided insights into the mechanisms of selenoprotein evolution. Characterization of selenoproteomes allows interpretation of other UGA codons in completed genomes of prokaryotes as terminators, addressing the UGA dual-function problem.     

Selective restoration of the selenoprotein population in a mouse hepatocyte selenoproteinless background with different mutant selenocysteine tRNAs lacking Um34

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA([Ser]Sec) gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T-->A) or 37 (37A-->G) in tRNA([Ser]Sec). The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2'-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N(6)-isopentenyladenosine. Both mutant tRNAs lacked the 2'-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with (75)Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.     

High-level expression in Escherichia coli of selenocysteine-containing rat thioredoxin reductase utilizing gene fusions with engineered bacterial-type SECIS elements and co-expression with the selA, selB and selC genes.

Mammalian thioredoxin reductase (TrxR) catalyzes reduction of thioredoxin and many other substrates, and is a central enzyme for cell proliferation and thiol redox control. The enzyme is a selenoprotein and can therefore, like all other mammalian selenoproteins, not be directly expressed in Escherichia coli, since selenocysteine-containing proteins are synthesized by a highly species-specific translation machinery. This machinery involves a secondary structure, SECIS element, in the selenoprotein-encoding mRNA, directing selenocysteine insertion at the position of an opal (UGA) codon, normally conferring termination of translation. It is species-specific structural features and positions in the selenoprotein mRNA of the SECIS elements that hitherto have hampered heterologous production of recombinant selenoproteins. We have discovered, however, that rat TrxR can be expressed in E. coli by fusing its open reading frame with the SECIS element of the bacterial selenoprotein formate dehydrogenase H. A variant of the SECIS element designed to encode the conserved carboxyterminal end of the enzyme (-Sec-Gly-COOH) and positioning parts of the SECIS element in the 3'-untranslated region was also functional. This finding revealed that the SECIS element in bacteria does not need to be translated for full function and it enabled expression of enzymatically active mammalian TrxR. The recombinant selenocysteine-containing TrxR was produced at dramatically higher levels than formate dehydrogenase O, the only endogenous selenoprotein expressed in E. coli under the conditions utilized, demonstrating a surprisingly high reserve capacity of the bacterial selenoprotein synthesis machinery under aerobic conditions. Co-expression with the selA, selB and selC genes (encoding selenocysteine synthase, SELB and tRNA(Sec), respectively) further increased the efficiency of the selenoprotein production and thereby also increased the specific activity of the recombinant TrxR to about 25 % of the native enzyme, with as much as 20 mg produced per liter of culture. These results show that with the strategy utilized here, the capacity of selenoprotein synthesis in E. coli is more than sufficient for making possible the use of the bacteria for production of recombinant selenoproteins.     

An algorithm for identification of bacterial selenocysteine insertion sequence elements and selenoprotein genes

MOTIVATION: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking. RESULTS: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes and predicted several new selenoprotein genes. Further analysis revealed that the size of bacterial selenoproteomes varied from 1 to 11 selenoproteins. Formate dehydrogenase was present in most selenoproteomes, often as the only selenoprotein family, whereas the occurrence of other selenoproteins was limited. The availability of the bacterial SECIS consensus and the tool for identification of these structures should help in correct annotation of selenoprotein genes and characterization of bacterial selenoproteomes.     

Selenocysteine insertion directed by the 3'-UTR SECIS element in Escherichia coli

Co-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria.     

A model for Sec incorporation with the regions upstream of the UGA Sec codon to play a key role

For eukaryotic selenoprotein mRNAs, it has been proposed that the SECIS element in the 3'-UTR is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still being considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human GPx mRNA. We found a protein, prepared from bovine brain microsomes, which strongly bound to the RNA fragment upstream of the UGA Sec codon but not to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. We also obtained the similar results with the RNA fragments of type I iodothyronine 5'-deiodinase (5'DI) mRNAs. Comparison of such RNA fragments with SECIS fragments revealed similarities in the region upstream of the in-frame UGA Sec codon of several Se-protein mRNAs. The study thus favors a novel model of Sec incorporation at the UGA Sec codon that involves the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF.     

Selenocysteine insertion sequence binding protein 2L is implicated as a novel post-transcriptional regulator of selenoprotein expression

The amino acid selenocysteine (Sec) is encoded by UGA codons. Recoding of UGA from stop to Sec requires a Sec insertion sequence (SECIS) element in the 3' UTR of selenoprotein mRNAs. SECIS binding protein 2 (SBP2) binds the SECIS element and is essential for Sec incorporation into the nascent peptide. SBP2-like (SBP2L) is a paralogue of SBP2 in vertebrates and is the only SECIS binding protein in some invertebrates where it likely directs Sec incorporation. However, vertebrate SBP2L does not promote Sec incorporation in in vitro assays. Here we present a comparative analysis of SBP2 and SBP2L SECIS binding properties and demonstrate that its inability to promote Sec incorporation is not due to lower SECIS affinity but likely due to lack of a SECIS dependent domain association that is found in SBP2. Interestingly, however, we find that an invertebrate version of SBP2L is fully competent for Sec incorporation in vitro. Additionally, we present the first evidence that SBP2L interacts with selenoprotein mRNAs in mammalian cells, thereby implying a role in selenoprotein expression.     

A regulatory role for Sec tRNA[Ser]Sec in selenoprotein synthesis

Selenium is biologically active through the functions of selenoproteins that contain the amino acid selenocysteine. This amino acid is translated in response to in-frame UGA codons in mRNAs that include a SECIS element in its 3' untranslated region, and this process requires a unique tRNA, referred to as tRNA([Ser]Sec). The translation of UGA as selenocysteine, rather than its use as a termination signal, is a candidate restriction point for the regulation of selenoprotein synthesis by selenium. A specialized reporter construct was used that permits the evaluation of SECIS-directed UGA translation to examine mechanisms of the regulation of selenoprotein translation. Using SECIS elements from five different selenoprotein mRNAs, UGA translation was quantified in response to selenium supplementation and alterations in tRNA([Ser]Sec) levels and isoform distributions. Although each of the evaluated SECIS elements exhibited differences in their baseline activities, each was stimulated to a similar extent by increased selenium or tRNA([Ser]Sec) levels and was inhibited by diminished levels of the methylated isoform of tRNA([Ser]Sec) achieved using a dominant-negative acting mutant tRNA([Ser]Sec). tRNA([Ser]Sec) was found to be limiting for UGA translation under conditions of high selenoprotein mRNA in both a transient reporter assay and in cells with elevated GPx-1 mRNA. This and data indicating increased amounts of the methylated isoform of tRNA([Ser]Sec) during selenoprotein translation indicate that it is this isoform that is translationally active and that selenium-induced tRNA methylation is a mechanism of regulation of the synthesis of selenoproteins.     

Intein-based Design Expands Diversity of Selenocysteine Reporters

The presence of selenocysteine in a protein confers many unique properties that make the production of recombinant selenoproteins desirable. Targeted incorporation of Sec into a protein of choice is possible by exploiting elongation factor Tu-dependent reassignment of UAG codons, a strategy that has been continuously improved by a variety of means. Improving selenoprotein yield by directed evolution requires selection and screening markers that are titratable, have a high dynamic range, enable high-throughput screening, and can discriminate against nonspecific UAG decoding. Current screening techniques are limited to a handful of reporters where a cysteine (Cys) or Sec residue normally affords activity. Unfortunately, these existing Cys/Sec-dependent reporters lack the dynamic range of more ubiquitous reporters or suffer from other limitations. Here we present a versatile strategy to adapt established reporters for specific Sec incorporation. Inteins are intervening polypeptides that splice themselves from the precursor protein in an autocatalytic splicing reaction. Using an intein that relies exclusively on Sec for splicing, we show that this intein cassette can be placed in-frame within selection and screening markers, affording reporter activity only upon successful intein splicing. Furthermore, because functional splicing can only occur when a catalytic Sec is present, the amount of synthesized reporter directly measures UAG-directed Sec incorporation. Importantly, we show that results obtained with intein-containing reporters are comparable to the Sec incorporation levels determined by mass spectrometry of isolated recombinant selenoproteins. This result validates the use of these intein-containing reporters to screen for evolved components of a translation system yielding increased selenoprotein amounts.     

SBP,SECIS binding protein,binds to the RNA fragment upstream of the Sec UGA codon in glutathione peroxidase mRNA

In mammals, most of the selenium contained in the body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is typically recognized as a translation stop signal, it is intriguing how a cell recognizes and distinguishes a UGA Sec codon from a UGA stop codon. For eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated the Sec insertion sequence (SECIS) in the 3'-untranslated (3'-UTR) region is required for recognition of UGA as a Sec codon. Some proteins which bind to SECIS (SBP) have been reported. However, it is not clear how the SECIS element in the 3'-UTR can mediate Sec insertion far at the in-frame UGA Sec codons. The idea that there must be a signal near the UGA Sec codon is still considered. Therefore, we searched for a protein which binds to an RNA sequence surrounding the UGA Sec codon on human glutathione peroxidase (GPx) mRNA. We found a protein which strongly bound to the RNA fragment upstream of the UGA Sec codon. However, this protein did not bind to the RNA sequence downstream of the UGA codon. This protein also bound to the SECIS sequence in the 3'-UTR of human GPx, and this binding to SECIS was competed with the RNA fragment upstream of the UGA Sec codon. Comparison of the RNA fragment with the SECIS fragment identified the conserved regions, which appeared in the region upstream of the in-frame UGA Sec codon of Se-protein mRNAs. Thus, this study proposes a novel model to understand the mechanisms of Sec incorporation at the UGA Sec codon, especially the regions upstream of the UGA codon of mRNAs of mammalian selenoproteins. This model explains that the stem-loop structure covering the UGA codon is recognized by SBP and how the UGA Sec codon escapes from attack by eRF of the peptide releasing factor.     

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.