How Selenium Has Altered Our Understanding of the Genetic Code

Dolph L. Hatfield 1* and Vadim N. Gladyshev 2

Molecular Biology of Selenium Section, Basic Research Laboratory,
Center for Cancer Research, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland 20892,, 1 Department of
Biochemistry, University of Nebraska, Lincoln, Nebraska 68588 2

Selenium is an essential micronutrient in the diet of many life
forms, including humans and other mammals. Significant health benefits
have been attributed to this element. It is rapidly becoming recognized
as one of the more promising cancer chemopreventive agents (19), and there are strong indications that it has a role in reducing viral expression (4), in preventing heart disease and other cardiovascular and muscle disorders (23), and in delaying the progression of AIDS in human immunodeficiency virus-infected patients (3). Additional evidence suggests that selenium may have a role in mammalian development (51), in immune function (70), in male reproduction (30), and in slowing the aging process (70).

Despite the many potential health benefits of selenium, the means by
which this element promotes better health are only just beginning to be
elucidated (31, 91). There are about 20 known selenium-containing proteins in mammals (33),
and it would seem very likely that several of these are mediators of
health benefits of dietary selenium. Therefore, it is critical to
understand how selenium is inserted into protein and the identities and
functions of the resulting protein products. Selenium is present in
naturally occurring selenium-containing proteins in two basic forms. It
can be inserted posttranslationally as a dissociable cofactor (32).
This rare form of protein-associated selenium has been found only in
several bacterial molybdenum-containing enzymes and will not be
discussed further in this review. Selenium is also cotranslationally
inserted into protein as the amino acid selenocysteine (Sec). Such
occurrence of this element in protein is widespread in all major
domains of life and is responsible for the majority of biological
effects of selenium. The elucidation of how Sec is incorporated into
protein has progressed at a rapid pace in the last decade and has
revealed some surprising results. In fact, unraveling this mystery has
altered our understanding of the genetic code, as the code has now been
expanded to include Sec as the 21st naturally occurring amino acid.
When the code was deciphered in the mid-1960s (48, 79),
20 amino acids were assigned to 61 of the possible 64 codons within the
triplet code and 3 codons were found to function as terminators for
protein synthesis. Each of the 64 code words was therefore assigned a
function, and there did not appear to be room for additional amino
acids. Although it was recognized in the mid-1960s that one code word,
AUG, had a dual role of initiating protein synthesis and inserting
methionine at internal protein positions, the possibility that a second
codon also had two functions was not considered at that time. We now
know that UGA serves as both a termination codon and a Sec codon. The
means by which UGA serves as a Sec codon and how Sec is biosynthesized
and incorporated into protein have been examined in considerable detail
with eubacteria (reviewed in reference 7)
and with mammals (this review). While the fundamental mechanism of Sec
insertion in these organisms appears to be similar, recent studies
suggest that mammals evolved additional components that allow
incorporation of multiple Secs into a single protein and provide
stringent regulation of Sec biosynthesis. The present review discusses
our current knowledge of these features in mammals.

It should be noted that selenium can also be incorporated nonspecifically into protein (42).
The nonspecific occurrence of this element in protein arises when
selenium replaces sulfur in the biosynthesis of cysteine or methionine
and the resulting selenoamino acid (Sec or selenomethionine) is
inserted in place of the natural amino acid. Such misincorporation of
selenium into protein may be toxic; this subject has been reviewed
elsewhere (42).

UGA DICTATES Sec INCORPORATION INTO PROTEIN

The initial studies, which suggested that UGA coded for Sec,
involved sequencing selenoprotein genes and aligning their open reading
frames with the amino acid sequences of the corresponding gene
products. The genes for two Sec-containing proteins, glutathione
peroxidase 1 (GPX1) in mammals (13, 77, 85) and formate dehydrogenase in Escherichia coli (103),
were the first genes found to contain TGA in the open reading frame.
Interestingly, the TGA codons aligned with the Sec residue in the
corresponding gene products. Such studies did not demonstrate
unequivocally that UGA codes for Sec. Theoretically, a tRNA decoding
UGA could introduce a precursor of Sec into the nascent selenopeptide
in response to UGA and the resulting amino acid residue would be
modified to Sec posttranslationally. Since Sec, unlike the other 20
amino acids in the genetic code, was found to be biosynthesized on its
tRNA (8, 41), the insertion of a precursor amino acid (e.g., phosphoserine [39
and references therein]) was a possibility. However, the fact that Sec
was subsequently s hown to be attached to its tRNA intracellularly in
both bacterial (59) and mammalian (55)
cells provided the strongest evidence at that time that Sec was indeed
the 21st amino acid in the genetic code. The expanded genetic code that
includes Sec is s hown in Fig. 1.

Sec tRNA, A MOST NOVEL tRNA

Sec tRNA (hereafter designated Sec tRNA [Ser]Sec and defined below
in “Sec biosynthesis occurs on its tRNA”) is the only known tRNA that
governs the expression of an entire class of proteins, the
selenoproteins. Sec tRNA [Ser]Sec has therefore been called the key
molecule (8) and the central component (41) in selenoprotein biosynthesis. The structure of Sec tRNA [Ser]Sec from mammals is s hown in Fig. 2 in both a 9/4 (8) and a 7/5 (24)
cloverleaf form (i.e., nine or seven paired bases in the acceptor stem
and f our or five paired bases in the T stem). Evidence for both
secondary structures has been presented (46, 47).
Sec tRNA [Ser]Sec has additional features that distinguish it from all
other tRNAs. For example, at 90 nucleotides in length, it is the
longest eukaryotic tRNA sequenced to date (2, 24, 26, 39). This is due to an atypically long variable arm and the presence of 13 nucleotides in the acceptor and T{Psi}C stem helices (where {Psi}indicates
pseud ouridine) instead of the 12 normally found in all other tRNAs.
Sec tRNA [Ser]Sec contains relatively few modified nucleotides (Fig. 2)
compared to other tRNAs, which may have as many as 15 to 17 modified
nucleotides. It may have up to six base pairs in the dihydr ouracil
stem instead of the three to f our found in other tRNAs. Transcription
of Sec tRNA [Ser]Sec is also unusual as it begins at the first
nucleotide within the coding sequence (56)
while all other tRNAs, whether they are of nuclear or organelle origin,
have a 5′ leader sequence that must be processed. Sec tRNA [Ser]Sec,
therefore, has a 5′ triphosphate on its terminal guanosine moiety. Many
additional novel features of Sec tRNA [Ser]Sec transcription have also
been reported, and this subject has been thoroughly reviewed elsewhere (42).

The Sec tRNA [Ser]Sec gene occurs in single copy in the genomes of
all mammals examined thus far, including humans, mice, rats, rabbits,
cattle, and Chinese hamsters (reference 10
and references therein). The primary sequence of the gene has also been
determined in chickens, frogs, zebra fish, fruit flies, and nematodes (10);
like that in mammals, it is 87 nucleotides long (the CCA terminus is
added posttranscriptionally to make its final length of 90
nucleotides). The only exception to the occurrence of single-gene copy
in the genomes of animals was found in zebra fish, where two gene
copies exit (100). The genomes of humans and rabbits also encode one pseudogene, while that of Chinese hamsters encodes three pseudogenes.

The Sec tRNA [Ser]Sec population in mammalian cells contains two
major isoforms that differ from each other by a single methyl group in
the wobble position (position 34) of the anticodon (see Fig. 2).
One isoform contains methylcarboxymethyl-5′-uridine (mcm 5U) at
position 34, and the other contains methylcarboxymethyl-5′-uridine-2′-O-methylribose (2, 26). mcm 5U is the precursor of mcm 5Um (17, 50),
and addition of this methyl group marks the final step in Sec tRNA
[Ser]Sec maturation. Interestingly, the addition of this methyl group
is responsive to selenium status (15, 26, 40), and its presence confers dramatic changes in tertiary structure (26). Efficient methylation of mcm 5U to form mcm 5Um requires the prior synthesis of each modified base (m 1A at position 58, {Psi}at position 55, and isopentenyladenosine [i 6A] at position 37) and an intact tertiary structure (50). Synthesis of m 1A, {Psi},
i 6A, and mcm 5U was not connected to primary and tertiary structure as
stringently as that of mcm 5Um. These studies, as well as those
demonstrating increased methylation at the 2′-O-ribose position in the presence of selenium (15, 26, 40) and alteration in tertiary structure with 2′-O-ribose methylation (26, 50), suggest that the two major Sec tRNA [Ser]Sec isoforms have different physiological roles.

As noted above, the distributions and relative amounts of mcm 5U and
mcm 5Um are influenced by selenium status, and their levels vary in
different mammalian cells and tissues (15, 26, 40).
The enrichment of the Sec tRNA [Ser]Sec population in the presence of
selenium is apparently due to reduced turnover rather than enhanced
transcription, as evidenced by studies s howing this effect in Xenopus oocytes in the absence of de novo transcription (17)
and by direct measurement of the effects of selenium on Sec tRNA
[Ser]Sec turnover in CHO cells (R. J. Coppinger, B. A. Carlson, M.
Butz, K. Esser, D. L. Hatfield, and A. M. Diamond, unpublished data).

Sec BIOSYNTHESIS OCCURS ON ITS tRNA

Although the biosyntheses of glutamine and asparagine can occur on
their tRNAs, this mode of synthesizing amino acids is restricted to
certain life forms and is not universal in nature (93).
In contrast, the biosynthesis of Sec that is incorporated into protein
in response to UGA codons is distinctive from the other 20 amino acids
in the genetic code in that its synthesis always occurs on its tRNA (7, 10).
Since Sec can be attached to tRNA Cys by cysteyl tRNA synthetase and
incorporated nonspecifically into protein in response to Cys codons (42),
then evolution of Sec biosynthesis on its tRNA provides another means
by which this amino acid could have been included in the genetic code.
This proposal is consistent with the suggestion that the presence of
Sec in the code occurred relatively late in the code’s evolution (33).

Sec tRNA [Ser]Sec is initially aminoacylated with serine in both prokaryotes (7) and eukaryotes (10), and serine serves as the backbone for Sec synthesis (7, 10, 90).
Since serine is attached to Sec tRNA [Ser]Sec by seryl tRNA synthetase
and the identity elements in Sec tRNA [Ser]Sec are for serine and not
Sec, but the amino acid inserted into protein is Sec, this tRNA has
been designated Sec tRNA [Ser]Sec (41).
The identity elements for mammalian Sec tRNA [Ser]Sec include the long
variable arm and the discriminator base, both of which are essential
for aminoacylation (80, 99). The acceptor, T{Psi}C, and D stems also play a role in the identity process (1).

The biosynthesis of Sec from serine on Sec tRNA [Ser]Sec has been completely characterized for E. coli (7, 8), but the specific steps in this process in mammals are unknown. In E. coli,
a pyridoxal phosphate-dependent Sec synt hase catalyzes the removal of
hydroxyl group from serine to form an aminoacrylyl intermediate. This
intermediate serves as the acceptor for activated selenium, resulting
in the formation of selenocysteyl- tRNA [Ser]Sec (7, 8). In mammals, a minor seryl tRNA which decoded UGA (38) and formed phosphoseryl tRNA (reference 39 and references therein) was subsequently identified as Sec tRNA [Ser]Sec (55).
The roles of the kinase and phosphoseryl tRNA [Ser]Ser in the
biosynthesis of Sec have not been characterized. However, the formation
of phosphoserine is consistent with a Sec synt hase-catalyzed reaction,
as phosphorylated serine would have a better leaving group than serine
in the Sec biosynthetic pathway.

The active form of selenium that is donated to the intermediate in
Sec biosynthesis has been identified in prokaryotes as
monoselenophosphate, which is synthesized from selenide and ATP by
selenophosphate synthetase (34). Although the active selenium donor in eukaryotes has not been characterized, it is likely the same selenium form (37, 49, 63). Two selenophosphate synthetase genes in mammals, designated Sps1 and Sps2, have been identified (37, 49, 63). SPS2 is a selenoprotein, suggesting that it is involved in the autoregulation of its own biosynthesis (37). Once the activated form of selenium is donated to the intermediate, the biosynthesis of Sec on tRNA [Ser]Sec is completed.

NOVEL INSERTION OF Sec INTO PROTEIN

The fact that UGA has a dual role of serving as a stop and a Sec codon (see Fig. 1)
raises an important question of how the cell distinguishes between
these two functions. Besides Sec tRNA [Ser]Sec and the in-frame UGA
codon in selenoprotein mRNA, there are several other factors that are
required for the donation of Sec to protein and dictate the specific
function of UGA as Sec. These include (i) the cis-acting stem-loop structure, designated the Sec insertion sequence (SECIS) element (62); (ii) the SECIS-binding protein 2 (SBP2) (21, 22, 64); and (iii) the Sec-specific elongation factor (EFsec, also called mSelB) (27, 92).

SECIS elements. SECIS elements are present in 3′ untranslated regions (3′-UTRs) of all eukaryotic selenoprotein genes (62).
In archaea, SECIS elements are also located in the 3′-UTRs, but the
structures themselves are different from the eukaryotic counterparts (82).
Bacterial SECIS elements differ from both eukaryotic and archaeal
structures and are located in the coding regions of selenoprotein
genes, immediately downstream of Sec-encoding UGA codons (7).

Eukaryotic SECIS elements are composed of two helixes separated by
an internal loop; a SECIS core structure, Quartet, located at the base
of helix 2; and an apical loop (Fig. 3).
The Quartet is formed by f our non-Watson-Crick interacting base pairs
and is the main functional site of the stem-loop structure (94).
When the apical loop is large enough, an additional ministem is formed
that presumably stabilizes the SECIS element. The presence of this
ministem was used to classify SECIS elements into form 1 and form 2
structures, with form 1 SECIS elements lacking, and form 2 SECIS
elements containing, the ministem (35).
These SECIS forms are interconvertible by mutations that extend or
shorten the apical loop or by natural evolution of selenoprotein genes.
Primary sequence conservation of eukaryotic SECIS elements is almost
nonexistent, with the only strictly conserved nucleotides being UGA in
the 5′ portion and GA in the 3′ portion of the Quartet. In addition, a
nucleotide immediately preceding the Quartet and two unpaired
nucleotides in the apical loop are adenosines in the majority of
selenoprotein genes (52).

The presence of a SECIS element in the 3′-UTR of selenoprotein genes
dictates any in-frame TGA codon within the coding region to serve as
Sec when a minimal spacing requirement between TGA and SECIS element
(51 to 111 nucleotides) is met (62).
This property suggests that SECIS elements are both necessary and
sufficient for Sec insertion, provided that mRNA bearing an in-frame
UGA and a 3′-UTR SECIS element have access to ribosome-based protein
synthesis machinery and Sec-specific translation factors. This property
also suggests that designing UGA-SECIS pairs in nucleotide sequences
can be used for targeted insertion of Sec into protein.

SBP2 and EFsec. SECIS elements function by recruiting SBP2 to form a tight SECIS-SBP2 complex (Fig. 4) (21, 22, 64). SBP2 binds to the SECIS Quartet and also to sequences directly preceding the Quartet, but not to the apical loop (21, 29).
The reason for strict conservation of the length of helix 2 (located
between the Quartet and adenosines in the apical loop) is not
understood. Evidence was also presented that SBP2 is stably associated
with ribosomes via 28S rRNA in a manner independent of its Sec
insertion function, suggesting that SBP2 preselects ribosomes for Sec
insertion (21).
An RNA-binding domain was identified in the C-terminal sequence of
SBP2, and an additional domain was identified that was required for Sec
insertion, but not for SECIS binding.

Besides binding to SECIS elements and ribosomes, SBP2 binds EFsec,
which in turn recruits Sec tRNA [Ser]Sec and inserts Sec into nascent
polypeptides in response to UGA codons (27, 92).
EFsec is specific for Sec and is different from EF1A, which is involved
in insertion of the other 20 amino acids. SBP2 and EFsec jointly
constitute the functional equivalent of the single SELB factor in
bacteria (7).
The occurrence of SBP2 and EFsec as separate proteins in eukaryotes
suggests a mechanism for rapid exchange of the Sec tRNA [Ser]Sec-EFsec
complex (from empty to aminoacyl tRNA bound), following Sec insertion.

Other factors. Additional trans-acting factors have been
implicated in Sec insertion in eukaryotes. Among these, Sec synt hase
is probably the major missing piece in the eukaryotic selenoprotein
machinery. Bacterial Sec synt hase was described several years ago
(reference 7
and references therein), but its counterpart in archaea and eukaryotes
is not known. As discussed above, the roles of the seryl tRNA [Ser]Sec
kinase and phosphoseryl tRNA [Ser]Sec in Sec biosynthesis are not
known, but characterization of the kinase would certainly shed light on
this issue. As also discussed above, Sec tRNA [Ser]Sec exists in two
isoforms that differ by a single methyl group, and the Sec tRNA
[Ser]Sec methylase may in addition play a role in regulating the
mammalian Sec insertion machinery.

UGA: STOP OR GO?

Even though we have considerable insight into the factors involved
in Sec insertion into protein (see above), there are several additional
aspects of selenoprotein biosynthesis that determine whether a UGA Sec
codon dictates termination or readthrough. Sec insertion appears to be
an inefficient process (references 28, 29, 64, and 66
and references therein), and clearly some Sec UGA codons support both
readthrough and termination. Selenoprotein P (SelP) from rat plasma
contains 10 Sec residues and occurs in f our isoforms (65).
The shorter isoforms arise from termination at the second, third, and
seventh UGA codons. Thus, these UGA codons are programmed to dictate a
cessation in SelP expression as well as a continuation in SelP
production. What then determines the fate of a UGA Sec codon? In
addition to the cis-elements UGA and SECIS that are essential for Sec insertion, there are several trans-acting factors as well as other cis-elements that influence the interplay between Sec incorporation and translation termination.

The nucleotide context of UGA is a cis-feature that also has a role in governing Sec incorporation versus translation termination (62, 69).
In mammals, a purine at the position immediately 3′ to UGA (the +1
position) favors termination, while a pyrimidine in this position
favors readthrough (62, 69). The base at the +2 position and the first codon (36) or the first two codons (78)
immediately upstream of UGA also influence termination efficiency. A +1
pyrimidine followed by a +2 purine appears to favor termination (36); interestingly, all 10 residues in rat plasma SelP contain either a +1 purine or the +1, +2 pyrimidine-purine combination (65).
It is not clear what role the penultimate codon or codons play in
termination versus readthrough in SelP, but it would seem that all 10
Sec residues are encoded in favorable termination contexts.
Furthermore, Ma et al. (65) suggested that trans-acting
factors likely play an important role in the fate of SelP UGA Sec
codons. It should be noted, however, that the +1 base and other
downstream cis-acting elements have been reported to play only a minor role in termination in one study (78) but a more significant role in another study (36).
The discrepancies in these two studies most certainly reflect the use
of different model (reporter) systems. Different Sec codons manifest
different insertion efficiencies (36, 65, 78) and thus may have different cis- and trans-acting requirements in establishing Sec insertion-translation termination interplay.

trans-Acting factors, such as SBP2, EFsec, Sec tRNA
[Ser]Sec, the termination factor eukaryotic released factor 1 (eRF1),
and the eRF1- and ribosome-dependent GTPase eRF3, are likely candidates
that regulate Sec incorporation-translation termination interplay (21, 29, 36, 64, 78). SBP2 is envisioned to function in Sec incorporation and to prevent translation termination (20, 21), and although this is most certainly the case, hard data supporting this model are lacking (21).
As discussed above, SBP2 is bound tightly to ribosomes and this and
other characteristics of SBP2 suggest that this factor is involved in
ribosome selection for Sec incorporation (21).
In this model, ribosomes containing SBP2 would incorporate Sec, while
those that do not would terminate translation. If SBP2 is first bound
to ribosomes, then a likely next step would involve the formation of a
quaternary complex between SBP2, the SECIS element, EFsec, and Sec tRNA
[Ser]Sec (21).
What is so intriguing about this model is that SBP2 is the major player
in determining the efficiency of selenoprotein synthesis.

Overexpressing selenoprotein mRNA either in vitro or in transformed
cells provides a model system for determining what factors are limiting
in selenoprotein expression, as the level of termination at the Sec UGA
codon increases due to limitation of one or more of the trans-acting factors (reference 64
and references therein). Under these conditions, SBP2 enhances
selenoprotein expression, whereas EFsec and Sec tRNA [Ser]Sec have only
marginal effects, demonstrating that SBP2 is the limiting factor. SBP2
is also limiting in rabbit recticulocytes (21).
Polysome loading onto mRNAs programmed for Sec incorporation was
decreased in wild-type compared to cysteine mutant mRNAs where UGA is
changed to a Cys codon (28, 66). However, polysome loading and Sec incorporation were increased by excess eRF1 (66) or SBP2 (28).
These results suggest a defect in translation at the UGA codon in
selenoprotein mRNAs and that this flaw in protein synthesis is
influenced by trans-acting factors.

Once SPB2 is bound to a SECIS element, it does not readily disassociate from the element (21, 64),
suggesting that SBP2 remains attached primarily to its element
throughout protein synthesis. Increased levels of eRF1, however, were
observed to have minimal affects on Sec incorporation (78) or to enhance this process (36), while excess eRF3 had no affect (36). Since it is envisioned that Sec incorporation competes with translation termination (28, 29, 64, 66) and that different UGA codons have different Sec incorporation efficiencies (36, 65, 78),
it was suggested that the termination factors are at saturating levels
intracellularly, and thus excess amounts of these components do not
have any apparent effect on Sec incorporation (78). Alternatively, excess RF1 may be involved in sequestering RF3 and therefore not be available for UGA Sec codon competition (36).
As noted above, the variations in findings reported in these two
studies are most likely due to the use of different model systems.

Overexpression of Sec tRNA [Ser]Sec was found to enhance Sec
insertion in one study involving an analysis of the factors effecting
Sec incorporation and translation termination (78). This finding, however, is not consistent with studies s howing that reductions (9, 14) or enrichments (75, 76)
in the levels of the Sec tRNA [Ser]Sec population in mammalian cells or
tissues do not affect selenoprotein biosynthesis. Inclusion of two or
three UGA codons in the same reading frame was found to result in
considerable reduction in synthesis of the full-length product compared
to that observed with a single UGA codon (78).
These investigators proposed, largely from this observation, that Sec
insertion favors a nonprocessive rather than a processive mechanism (78).

SELENOPROTEINS: IDENTITY AND FUNCTIONS

The known Sec-containing proteins in animals are s hown in Fig. 5.
The number of selenoproteins identified has increased dramatically in
the last several years. Interestingly, with the exception of
selenophosphate synthetase, there is no overlap between eukaryotic and
prokaryotic selenoproteomes (all selenoproteins in an organism).
Bacterial and archaeal selenoproteins are primarily involved in
catabolic processes and utilize selenium to catalyze various redox
reactions (84).
In contrast, functionally characterized eukaryotic selenoproteins
participate in antioxidant and anabolic processes. These observations
suggest an independent origin of prokaryotic and eukaryotic
selenoproteomes (33).

FIG. 5. Animal Sec-containing proteins. All currently known selenoproteins
are listed (left). The relative sizes of selenoproteins (empty boxes)
and the locations of Sec (red box) and an {alpha}-helix immediately downstream of Sec (green box) in the selenoprotein sequences are indicated (right).

In eukaryotes, disruption of the Sec tRNA [Ser]Sec gene
is embryonically lethal, suggesting an essential function for one or
more selenoproteins in development (9).
One candidate for an essential selenoprotein gene is the thioredoxin
reductase gene. The protein expressed by this gene is present in all
living organisms, but its Sec-containing form occurs only in animals.
Moreover, disruption of thioredoxin, a substrate for thioredoxin
reductase, is lethal, as s hown by studying mice lacking the
thioredoxin gene (68).

One general theme is evident from the analysis of eukaryotic
selenoproteins. Although these selenoproteins do not have sequence
homology, similar structures, or related functions, the location of Sec
in these proteins appears to be limited to only several positions. In
fact, the majority of eukaryotic selenoproteins can be assigned to one
of two groups according to Sec location (31).
One selenoprotein group includes proteins containing Sec in the
N-terminal portions of short domains. These proteins are largely {alpha}ß proteins, and Sec is often located in these proteins in the loop between a ß-strand and an {alpha}-helix,
according to secondary structure predictions. This location is similar
to that of the CXXC motif (two cysteines separated by two other amino
acids), which is involved in the redox reactions catalyzed by
thiol-disulfide oxidoreductases. In fact, several selenoproteins employ
a similar redox motif, except that one of the Cys residues is replaced
by Sec. For example, SelW, SelT, SelM, BthD, and their homologs possess
a CXXU motif (where U is Sec), whereas SelP and its homologs have a
UXXC motif. Other selenoproteins of this group, such as the GPX
homologs, contain only a single Sec (i.e., no Cys partner in the CXXC
motif), suggesting that Sec forms either predicted intermolecular
selenosulfide bonds or selenenic acid derivatives during redox
catalysis.

The second group of eukaryotic selenoproteins is characterized by
the presence of Sec in C-terminal sequences. In three mammalian
thioredoxin reductases, which contain a C-terminal GCUG motif, Sec is
located in the flexible C-terminal extension (86).
This situation is functionally similar to the fusion of a
low-molecular-weight redox compound to the C terminus of a common
functional domain (in the case of thioredoxin reductases, it is a
pyridine nucleotide disulfide oxidoreductase domain) (87).
The function of the Sec-containing motif in thioredoxin reductase is to
transfer reducing equivalents from the buried disulfide active site to
the active center of a protein substrate. In the case of thioredoxin
reductase 2, which contains an additional N-terminal thiol-disulfide
oxidoreductase domain, the Sec center transfers electrons to this
domain (87). An additional protein of the C-terminal Sec group is the Drosophila melanogaster G-rich protein (67).
This protein also contains a C-terminal penultimate Sec residue,
followed by a C-terminal glycine. The function of the G-rich protein is
not known.

Independently of the location of Sec in functionally characterized
selenoproteins, this amino acid appears to participate in redox
reactions. In selenoenzymes where Sec has a close Cys partner (e.g.,
SelT, SelW, BthD, etc.), secondary structure appears to stabilize a
highly reactive selenolate, whereas in the positions close to the C
terminus, the advantage of Sec over Cys may be due to its lower pK a.
Indeed, most cysteines are protonated under physiological pH
conditions, whereas Sec residues (pK a, ~5.5)
are ionized. The role of steric differences has also been suggested to
account for the use of Sec. For example, the C terminus of animal
thioredoxin reductases, Gly-Cys-Sec-Gly, forms an intramolecular
selenosulfide bond (57, 102).
However, the corresponding disulfide bond is not stable due to the
decreased atomic size of sulfur compared to that of selenium.

Among functionally characterized mammalian antioxidant
selenoproteins are f our glutathione peroxidases and three thioredoxin
reductases. In addition, recent studies revealed that one of the new
selenoproteins, SelR, is a zinc-containing methionine sulfoxide
reductase with specificity for methionine-R-sulfoxides (54). MsrA, an enzyme catalyzing a complementary reaction (i.e., a methionine-S-sulfoxide reduction), has been known for decades (98). It has been implicated in antioxidant defense and the life span of mammals (74). With the discovery of SelR function, a possibility is raised that selenium is also involved in aging.

It should be noted that the functions of the majority of
selenoproteins are not known. Characterization of their functions is an
obvious direction in selenoprotein research.

HIERARCHY OF SELENOPROTEIN EXPRESSION

A characteristic of selenium deficiency in mammals is that a
hierarchy exists with respect to maintaining the levels of individual
selenoproteins and retaining selenium in different organs (5, 45, 58, 64, 72).
For example, in selenium-deficient rats, GPX1 activity was reduced to
1% of that observed in the livers of selenium-sufficient rats and to
about 4 to 9% in selenium-deficient heart, kidney, and lung tissue.
GPX4 activity, however, was reduced only to 25 to 50% in these tissues
and was unaffected in testes. Interestingly, transgenic mice expressing
i 6A-deficient Sec tRNA [Ser]Sec had reduced levels of selenium in
their tissues and a hierarchy of selenoprotein activities similar to
that observed with selenium-deficient mice (76). The extent of selenoprotein reduction varied in the i 6A-deficient Sec tRNA [Ser]Sec mice, depending on the organ examined (76).

During selenium deprivation in the diets of rats and mice, the
amounts of this element were substantially reduced in liver and kidney,
while brain and testes retained most of their selenium (5, 44). The levels and maturation of Sec tRNA [Ser]Sec (15, 26, 40) and the efficiency of selenoprotein synthesis (see references 21 and 36
and references therein) are responsive to selenium status; thus, these
parameters are more affected in liver and kidney than in brain and
testes by changes in selenium status (15, 26, 44).
The greater sensitivity of GPX1 activity to selenium deficiency has
been attributed largely to an increased turnover in mRNA (18, 58, 83).
The enhanced degradation of GPX1 mRNA under conditions of selenium
deficiency occurs by the surveillance pathway, designated
nonsense-mediated decay (NMD), where the UGA Sec codon is recognized as
nonsense (73, 89, 97).
Interestingly, the position of the UGA Sec codon relative to the sole,
downstream intron in GPX1 mRNA determines whether the mRNA is subject
to NMD (88).
However, other selenoprotein mRNAs, such as DI1, GPX4, and SelP, are
not as sensitive as GPX1 to NMD during selenium deprivation despite the
presence of introns downstream of their UGA codons (44, 58, 89).
The reduction in GPX1 activity in transgenic mice carrying i
6A-deficient Sec tRNA [Ser]Sec is not likely due to mRNA turnover,
since GPX1 mRNA levels were not significantly altered in the kidneys of
these animals compared to those of wild-type animals (76).
SBP2 has also been reported to preferentially recognize SECIS elements
in specific selenoprotein mRNAs, suggesting a mechanism to account, at
least in part, for selenoprotein expression hierarchy during selenium
deficiency (64).
However, another group found little or no difference in SPB2
recognition of the SECIS element and suggested that this is not likely
to be a mechanism involved in selenoprotein hierarchy (29).
Both groups agreed that if SPB2 recognition of SECIS elements is
involved in the extreme sensitivity of GPX1 mRNA to NMD, then an
additional factor must also be required. In any case, it would seem
that there are several levels of regulation involved in determining the
priority of selenoprotein synthesis under various biological
conditions.

IDENTITY OF Sec UGA CODONS

Since the occurrence of UGA in the genetic code is most commonly
used for the cessation of protein synthesis, the identification and
correct annotation of selenoprotein genes containing Sec-encoding UGAs
have been difficult. In fact, the absolute majority of selenoprotein
genes are incorrectly annotated in completely sequenced genomes,
including the human genome. Typically, Sec-encoding TGA codons are
recognized by the currently available annotation programs as stop
signals; alternatively, the entire exons containing TGA codons are not
recognized. In addition, there are examples when in-frame 5′-UTR
sequences or terminator UGA codons were incorrectly interpreted as Sec
codons (12).

Since selenoprotein genes do not have a common amino acid consensus
sequence or a functional motif and the location of TGA within coding
sequences is not universally conserved, the SECIS element provides an
identifier that can help in the annotation of uncharacterized
selenoprotein genes. Although conservation of the primary sequence of
SECIS elements is low, their secondary structures are conserved. In
addition, calculation of the free energy of SECIS elements as a measure
of their stability aided in describing these structures
computationally. A computer program, SECISearch, has been developed
that is capable of identifying selenoprotein genes in nucleotide
sequence databases (53).
Initial applications of SECISearch or similar approaches to expressed
sequence tag databases identified three selenoprotein genes and were
the first examples of identification of new genes by searching for RNA
structures (53, 60). Subsequently, this approach was applied to the entire genome of D. melanogaster (11, 67).
These studies revealed the presence of three selenoprotein genes,
including two proteins (G-rich protein and BthD) that had no homology
to known proteins (67).
Not surprising, these genes were incorrectly annotated in the completed
fly genome. Identification of selenoprotein genes through recognition
of SECIS elements should be useful in the future analysis of the human
genome.

CONSEQUENCES OF CHANGES IN Sec tRNA [Ser]Sec EXPRESSION

Since Sec tRNA [Ser]Sec is absolutely required for the expression of
a relatively small class of proteins, genetic manipulations of this
molecule can be used to study selenoproteins and the role of selenium
in essential biological processes. The consequences of both
overexpressing (75, 76) and underexpressing (9, 14)
Sec tRNA [Ser]Sec have been examined, as well as the consequences of
expressing different mutant Sec tRNA [Ser]Sec forms (reference 76 and see below). Chinese hamster ovary cells were transfected with varying numbers, up to as many as 10 (75),
of Sec tRNA [Ser]Sec gene copies and transgenic mice carrying as many
as 20 wild-type Sec tRNA [Ser]Sec transgenes were generated, but there
was no detectable effect on selenoprotein biosynthesis in either study.
Most of the increase in the amount of the Sec tRNA [Ser]Sec population
occurred in mcm 5U levels, suggesting that the methylase that converts
this isoform to mcm 5Um is limiting for tRNA maturation. The fact that
selenoprotein biosynthesis was not affected by enriching the Sec tRNA
[Ser]Sec population demonstrates that the Sec tRNA [Ser]Sec isoforms
are not limiting in protein synthesis.

The Sec tRNA [Ser]Sec population has also been reduced approximately
in half in mice that were heterozygous for a targeting vector lacking
the Sec tRNA [Ser]Sec gene (9) and in mouse embryonic stem cells that were heterozygous for a similar targeting vector (14). GPX1 levels were virtually the same in wild-type and heterozygous cultured cells (14) and in each of the tissues examined in wild-type and heterozygous mice (9),
suggesting that the Sec tRNA [Ser]Sec population is not limiting. As
discussed above, removal of both copies of the Sec tRNA [Ser]Sec gene
from the mouse genome is embryonically lethal, demonstrating that
selenoprotein expression is essential in mammalian development (9).

The consequences of overexpressing either a mutant Sec tRNA [Ser]Sec lacking the highly modified i 6A at position 37 (76)
or containing an A at position 34 in place of mcm 5U or mem 5Um (M.
Moustafa, B. Carlson, M. El-Saadani, M. Rao, and D. Hatfield,
unpublished data) on selenoprotein synthesis were examined by
introducing multiple copies of the corresponding mutant gene into the
mouse genome. The levels of several selenoproteins were altered in mice
expressing either mutant Sec tRNA [Ser]Sec in a protein- and
tissue-specific manner (reference 76
and unpublished data). Since the mRNA levels of those selenoproteins
that were most effected by expression of the i 6A – Sec tRNA [Ser]Sec
remained essentially the same, the defect in selenoprotein synthesis
occurred at the translation step. Maturation of Sec tRNA [Ser]Sec was
inhibited in both these transgenic strains, as evidenced by the
reduction in the mcm 5Um isoform. These studies mark the first examples
of transgenic mice engineered to encode functional tRNA transgenes and
provide a model system for studying the role of specific selenoproteins
in health.

UGA, A LOGICAL CODON CHOICE FOR Sec IN EVOLUTION

It can be argued that UGA is by far the most fascinating codon
within the genetic code as it likely has served more functions than any
other code word in evolution. For example, an examination of current
genetic language s hows that UGA functions as a termination codon (79); a Sec codon (8, 41); a cysteine codon in Euplotes octocarinatus (71); a tryptophan codon in mitochondria (81), Mycoplasma, and Sprioplasma (81, 95); an inefficiently read tryptophan codon in Bacillus subtilis (61); and an inefficiently read codon in E. coli that is presumably decoded by tryptophan tRNA (96). In mammals, the UGA stop codon in rabbit ß-globin mRNA has been s hown to serve as many as eight functions (16),
including a stop codon; a suppressor codon that supports partial
readthrough for Arg Cys, Trp, and Ser tRNAs (the latter tRNA is Sec
tRNA [Ser]Sec, which is aminoacylated with serine [16]);
and a translation reading gap codon with the abyss consisting of one,
two, or three codons. The fact that other globin mRNAs terminate in UAA
or UAG, but do not appear to serve as suppressor codons or to promote
translation reading gaps, suggest that these functions are associated
solely with UGA.

Since other stop or infrequently read codons can code for Sec when
the anticodon in Sec tRNA [Ser]Sec is complementary to the
corresponding codon used in place of TGA (6, 43),
it would seem that any of a number of codons could have evolved for
Sec. However, the variety of functions of UGA suggest that this codon
has been loosely programmed in evolution and therefore is the most
likely code word to have evolved for the infrequently used amino acid
Sec. This possibility would seem to be even more plausible if the
inclusion of Sec in the genetic code occurred in evolution after the
code had evolved rather than if the original code accommodated Sec.

It should be noted that there are two contrasting proposals about
when living organisms acquired the ability to synthesize selenoproteins
(8, 33).
One suggests that Sec was encoded by UGA in primitive anaerobic
organisms and was a component of the primordial genetic code (8).
In this theory, the subsequent increase of oxygen in the atmosphere by
photosynthetic organisms counterselected against the use of Sec,
because of the sensitivity of this amino acid to oxidation. An
alternative hypothesis posits that Sec evolved only in the later stages
of the development of the genetic code and that the number of
selenoproteins accumulated rather than decreased in evolution (33).
In contrast to the idea that a declining use of Sec occurred in
evolution, this latter proposal suggested that many eukaryotic
selenoproteins, serving as antioxidant and redox proteins, were
employed by aerobic organisms to function in antioxidant systems.

As discussed above, Sec is dramatically different from any other of
the 20 protein amino acids in the mode of its incorporation and basic
biosynthetic steps. It is the only amino acid that directly requires a
structural element in mRNA in addition to the information specified by
the genetic code. It is synthesized on its own tRNA, while free Sec is
not a substrate for selenoprotein synthesis. The Sec biosynthetic
machinery is strikingly different from that of other amino acids and
employs additional Sec-specific components. These unique features of
Sec biosynthesis and insertion favor the view that Sec was added to the
already existing genetic code to take advantage of the unique chemistry
of selenium to counteract environmental stress and/or evolve new
functions (33).

SUMMARY

The only addition of a new amino acid to the genetic code since this
code was deciphered in the mid 1960s was the inclusion of the
selenium-containing amino acid, Sec, that is coded by UGA. UGA,
therefore, functions as both a signal for termination and a codon for
Sec. Tremendous progress has been made in recent years in understanding
the mechanism of how Sec is synthesized and inserted into nascent
selenopeptides in mammals. This includes discoveries of how specific
3′-UTR mRNA structures, designated SECIS elements, function in
recruiting SBP2, the Sec-specific EF, and selenocysteyl-tRNA [Ser]Sec
into a large Sec insertion complex, the selenosome. In this unique
amino acid insertion system, Sec tRNA [Ser]Sec is the key molecule that
is used both as the site for Sec biosynthesis and for its incorporation
into protein. The gene carrying this tRNA has been used as a tool to
study the expression of selenoproteins by the introduction of
additional wild-type and mutant transgenes into the mouse genome and by
removal of the gene from the mouse genome. SECIS elements have been
used in computational screens to identify a number of new selenoprotein
genes, whose characterization will shed light on many biological and
health-related properties of selenium.

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