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Molecular Cell, Vol. 13, 157–168, January 30, 2004, Copyright 2004 by Cell Press
Reprogrammed Genetic Decoding
in Cellular Gene Expression
Olivier Namy,1,* Jean-Pierre Rousset,2
Sawsan Napthine,1 and Ian Brierley1
1
Division of Virology
Department of Pathology
University of Cambridge
Tennis Court Road
Cambridge, CB2 1QP
United Kingdom
2
Institut de Genetique et Microbiologie
Laboratoire de Genetique Moleculaire de la Traduction
Universite Paris-Sud
Bât 400, 91405 ORSAY cedex
France
Reprogrammed genetic decoding signals in mRNAs
productively overwrite the normal decoding rules of
translation. These “recoding” signals are associated
with sites of programmed ribosomal frameshifting,
hopping, termination codon suppression, and the incorporation of the unusual amino acids selenocysteine
and pyrrolysine. This review summarizes current knowledge of the structure and function of recoding signals
in cellular genes, the biological importance of recoding
in gene regulation, and ways to identify new recoded
genes.
Introduction
Postgenomic studies constitute one of the current challenges in biology due to the burgeoning quantity of genome sequence available and the difficulties in utilizing
the data to understand how an organism functions. A
major problem is to couple the DNA sequences with the
battery of proteins a given organism can express. One
step relates to the decoding of genetic information into
polypeptides by way of translation. The genetic code,
deciphered almost 50 years ago, provides the fundamental clues for such decoding. However, deducing the
protein(s) encoded by a given DNA segment remains a
difficult task, even in simple microorganisms with few
or no introns. This is due, in part, to our imperfect knowledge of the signals embedded in the genome that are
involved in the translation of the genetic information.
The latter is spectacularly illustrated for some genes,
where the standard rules of decoding are subverted by
“recoding” signals (Gesteland et al., 1992) that promote
alternative decoding events like programmed ribosomal
frameshifting, hopping, and termination codon reassignment (Atkins et al., 2001; Brierley and Pennell, 2001;
Stahl et al., 2001). Several recoding events have been
characterized in transposable elements and viruses, but
the recent advances in genomics opens avenues for
the discovery of related and novel recoding signals in
cellular genes. Comprehensive analysis of the genome
of Saccharomyces cerevisiae, for example, indicates
that frameshifting and stop codon readthrough could
*Correspondence: [email protected]
Review
be far more frequent events than previously suspected
(Harrison et al., 2002; Namy et al., 2002, 2003; Shah
et al., 2002). This is supported by the description of
biologically important recoding events in the genes of
several other eukaryotes and also in gram-positive,
gram-negative, and methanogenic bacteria. To date,
most of these have been identified serendipitously and
a systematic search for recoding sites in sequenced
genomes is urgently needed. Here, recoding in conventional cellular genes is reviewed, focusing on the biological importance of recoding in gene regulation. Approaches to identifying novel recoded genes are also
discussed.
Translational Recoding
The dogma of the universal genetic code has been compromised by the finding that in certain organisms and
organelles, the meaning of selected codons has been
changed; for example, the specification of serine by
CUG in Candida albicans (Santos et al., 1997) or of tryptophan by UGA in mitochondria (Barrell et al., 1979). In
addition to such reassignment, which affects all genes
of a given organism, alternative ways of reading the
genetic code have been described that are programmed
by signals present in specific mRNAs at defined locations in the messenger. Such recoding events occur
generally in competition with standard decoding and
allow the synthesis of two or more polypeptides, at a
defined ratio, from a single mRNA molecule. Recoding
can occur during translational elongation (⫹1 or –1
frameshifting, hopping) or at the termination step (stop
codon readthrough/redefinition) (Gesteland and Atkins,
1996; Baranov et al., 2002). In frameshifting, the ribosome is induced to shift to an alternative, overlapping
reading frame, whereas in hopping, the ribosome effectively detaches from the mRNA at a take off site until a
landing site is reached where peptidyl tRNA re-pairs
functionally to the mRNA and translation continues. In
readthrough, the specific context of the termination codon is such that occasionally it is decoded by a tRNA
rather than a release factor, allowing ribosomes to synthesize an extended polypeptide. Sometimes, this tRNA
specifies an unusual amino acid, selenocysteine (Hatfield and Gladyshev, 2002) or pyrrolysine (Hao et al.,
2002; Srinivasan et al., 2002). Recoding events also affect cell physiology in numerous ways and regulate a
range of cellular processes (Table 1).
Recoding during Translational Elongation
Natural frameshift errors occur very rarely, but programmed ribosomal frameshifting signals increase the
probability of tRNA slippage enormously, occasionally
to such an extent that up to 50% of ribosomes change
frame. The general principles of ⫹1 (3⬘-wards) and –1
(5⬘-wards) programmed frameshifting have largely been
elucidated from studies of viruses, retrotransposons,
and insertional elements (Chandler and Fayet, 1993; Atkins et al., 2001; Brierley and Pennell, 2001; Stahl et al.,
2001; Plant et al., 2003). In essence, frameshifting is
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Table 1. Recoding Sites in Cellular Genes
Translational Step
Recoding Type
Elongation
⫹1 frameshifting
⫺1 frameshifting
Hopping
Termination
Readthrough
Selenocysteine
Pyrrolysine
Gene(s)
Function
Species
prfB
oaz1, 2, 3
ABP140
EST3
IL-10
PKAR
EoNDR2
p43
TERT1, 2, 3
dnaX
cdd
HpfucT2
fucA1
Edr
plaA
CS3
topo-3
oaf
kelch
hdc
PDE2
⬎15
⬎40
MtmB1/B2
release factor
antizyme
actin reorganization
telomerase subunit
Immune response modulator
cAMP protein kinase
nuclear protein kinase
telomerase subunit
telomerase subunit
DNA polymerase III
cytidine deaminase
␣(1,2) fucosyltransferase
␣-fucosidase
Unknown
Adhesin
Pili formation
DNA topoisomerase 3
Neuronal development
Ring canal actin organizer
tracheal branching inhibitor
cAMP regulation
Redox reaction → prok
Antioxidant → euk
methylamine methyltransferase
prok
euk
euk
euk
euk
eup
eup
eup
eup
prok
prok
prok
prok
euk
prok
prok
prok
euk
euk
euk
euk
prok
euk
prok
Cellular genes employing a recoding strategy are detailed. The host species is identified by prok (prokaryote), euk (eukaryote), and eup
(euplotes). Further details and relevant references are provided in the text. Data can be found at http://recode.genetics.utah.edu/
triggered by two elements, a slippery sequence in the
mRNA, where tRNA movement or misalignment is favored, and a stimulator that enhances the process, probably by induction of a ribosomal pause. Often the stimulator is a slowly decoded region in the mRNA, like a
codon for a low-abundance tRNA or a stop codon. Alternatively, a local mRNA secondary structure is present,
a stem-loop or pseudoknot that may perturb normal
decoding beyond simple induction of a pause (Kontos
et al., 2001).
Programmed ⫹1 Frameshifting in Prokaryotes
The prfB gene of E. coli, encoding release factor 2 (RF2),
harbors the paradigm ⫹1 ribosomal frameshift signal,
yet remains the only example of this class in a prokaryotic gene (Craigen and Caskey, 1986). RF2 recognizes
and promotes translation termination at UAA and UGA.
The prfB open reading frame (ORF) is interrupted by a
UGA stop codon some 26 codons downstream of the
initiation codon, yet the coding sequence continues immediately, albeit in the ⫹1 reading frame, for a further
340 codons. The frameshift, typically by some 30%–50%
of ribosomes, occurs at the slippery sequence CUU UGA
C (the zero frame is indicated by triplets) present at the
junction of the zero and ⫹1 ORFs, and generates active
RF2 protein (Figure 1). Mechanistically, the process requires tRNALeu decoding zero frame CUU to move a
single base 3⬘-wards onto UUU in the ⫹1 frame, a process enhanced in three ways; the similar anticodon:
codon contacts formed postslippage, the relatively poor
termination context of the stop codon (UGAC) and the
stimulatory effect of base pairing of a Shine-Delgarno
(SD)-like sequence, situated 3 nt upstream of the slippery site, with the anti-SD sequence of 16S rRNA (Weiss
et al., 1988). The distance between this SD-like sequence
and the start of the RF2 slippery sequence is shorter than
that typically seen between the SD and the initiator AUG
(5 nt; Chen et al., 1994) and this may lead to some “tension”
in the mRNA, promoting mRNA realignment in the 3⬘ direction (a push forward). This recoding event represents
an elegant autoregulatory mechanism controlling the
abundance of RF2 (Adamski et al., 1993). At high RF2
levels, the competition between termination and frameshifting is shifted in favor of termination, leading to a
decrease of the RF2 concentration in the cell. At this
point, frameshifting begins to predominate, raising RF2
levels. The fine-tuning facilitated by regulating RF2 levels by frameshifting may be required to maintain a constant termination efficiency at UGA codons, particularly
for genes in which this codon is used to specify the
incorporation of selenocysteine into proteins (see readthrough section below). The selection of tRNA(Ser)Sec is in
competition with RF2 (Mansell et al., 2001) and if the
concentration of the release factor became too high it
could interfere with selenocysteine incorporation and
prevent selenoproteins from playing a key role in activities such as the catalysis of redox reactions.
Programmed ⫹1 Frameshifting in Eukaryotes
Several ⫹1 frameshift signals have been described in
eukaryotic mRNAs. Perhaps the most intricate is present
in the mRNA encoding ornithine decarboxylase antizyme (Matsufuji et al., 1995). Antizyme 1 (AZ1) regulates
the stability of ornithine decarboxylase (ODC), an enzyme responsible for converting ornithine to putrescine
(Figure 2A), by targeting it to the proteasome in an unusual ubiquitin-independent mechanism, after which it
gets recycled (Murakami et al., 1992). The AZ1 frameshift
site comprises a slippery sequence that includes and
requires the ORF1 stop codon (5⬘ UCC UGA U 3⬘), and
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159
Figure 1. Autoregulation of RF2 Synthesis by
⫹1 Frameshifting
The frameshift signal of the prfB gene is depicted, with the internal termination codon
(UGA) highlighted (STOP) and the number of
amino acids to the termination codon in the
⫹1 frame shown. As RF2 becomes limiting
(blue arrow) due to termination at this site,
⫹1 slippage of tRNALeu occurs from the zeroframe codon CUU (underlined) to the overlapping ⫹1 frame codon (UUU), enhanced by the
upstream SD-like sequence (purple). However, as RF2 accumulates (red arrow), translation termination begins to predominate.
two stimulatory elements; a core sequence of unknown
action (5⬘ CCG GGG CCU CGG 3⬘) upstream of the slippery sequence and an RNA pseudoknot beginning 3 nt
downstream of the ORF1 stop (Figure 2B). AZ1 activity
resides only in the frameshift product, but the precise
mechanism of frameshifting is not known. Like RF2,
AZ1 frameshifting is linked to a feedback mechanism,
although in this case as a sensor to regulate polyamine
levels in mammalian cells. At high concentrations, ⫹1
frameshifting is increased, promoting the synthesis of
AZ1, which in turn degrades ODC, leading to a reduction
in cellular polyamine concentrations (Ivanov et al., 2000).
Figure 2. Regulation of Cellular Polyamine Levels Using Antizyme ⫹1 Frameshifting as a Sensor
(A) High polyamine levels stimulate ⫹1 frameshifting required for the synthesis of functional antizyme 1 (AZ1). AZ1 binds ornithine decarboxylase
(ODC) and triggers its degradation by the 26S proteasome, being itself recycled. As ODC catalyzes the first step of the polyamine biosynthesis
pathway, its degradation leads to a decrease in polyamine levels, which in turn reduces frameshifting efficiency.
(B) Key features of the AZ1 frameshift signal are shown. The site of frameshifting is a “shifty stop” (5⬘ UCC UGA 3⬘, in blue and red, respectively)
and the process is enhanced both by a stimulatory RNA pseudoknot just 3⬘ of the slippery sequence and a poorly defined 5⬘ stimulatory
sequence (?). The mechanism of frameshifting is not known. Currently an occlusion model is favored, with the pseudoknot and/or upstream
stimulator modifying the structure of the A site (when occupied by the termination codon UGA) such that the U is occluded and the ⫹1
frame GAU codon can be decoded out-of-frame by tRNAAsp (Atkins et al., 2001). How polyamines stimulate the frameshift event remains to
be determined.
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160
Figure 3. Conservation of EST3 Organization among Saccharomyces Species
EST3 expression requires a ⫹1 frameshift to fuse the products of
ORF1 (93 amino acids) and ORF 2 (92 amino acids). The percentage
amino acid identity between the sequences identified in S. cerevisiae
and related Saccharomyces species is shown. The “missing” portion
of ORF2 in S. bayanus (*) reflects the absence of sequence information (contig. end). The slippery sequences identified in the EST3
homologs, which are identical, are indicated with the stop codon
of ORF1 in red.
Frameshifting is employed in the expression of all known
antizymes in many different species and in all cases to
date, the process is used as a sensor of free polyamines.
The conservation of this mechanism throughout evolution highlights a crucial role for frameshifting in the regulation of polyamine levels.
A requirement for ⫹1 frameshifting in telomerase activity has been shown in the last few years. The synthesis
of telomeres in S. cerevisiae, as in many organisms,
depends upon telomerase, a reverse transcriptase that
uses an internal RNA as a template. The yeast enzyme
is a ribonucleoprotein composed of at least four proteins
(Est1p, Est2p, Est3p, Cdc13p) and the template RNA
(TLC1). Est3p is a stable component of the telomerase
holoenzyme and essential for the maintenance of telomeres in vivo. The ⫹1 frameshifting mechanism employed in the expression of the protein involves an essential, short slippery sequence, CUU AGU U (Morris
and Lundblad, 1997). As the AGU codon in this stretch
is decoded by a low abundance tRNA, a ribosomal
pause is thought to occur, promoting ⫹1 slippage of
peptidyl tRNALeu from the CUU to the overlapping UUA
codon. Unsurprisingly, this slippery sequence is somewhat underrepresented in the S. cerevisiae genome (34
of 115 expected; Shah et al., 2002) but it is not especially
rare, raising the possibility that other flanking stimulators may yet be identified, or that other functional sites
exist. That frameshift efficiencies promoted by the slippery sequence alone (Shah et al., 2002) are lower than
those seen with the complete EST3 sequence (Morris
and Lundblad, 1997) supports the existence of such
elements. The organization of EST3 genes and the utilization of ⫹1 frameshifting are conserved among different Saccharomyces species suggesting a key role for
frameshifting in telomere maintenance (Figure 3).
Additional support for this belief comes from an analysis of the genome of the ciliate Euplotes. Ciliates have
long been known to employ unconventional decoding in
their reassignment of termination codons, with Euplotes
decoding the conventional stop codon UGA as cysteine
(Meyer et al., 1991). However, their capacity to use alternative genetic decoding can be extended to the utilization of ⫹1 frameshifting to express nuclear proteins (Klobutcher and Farabaugh, 2002). It has been estimated
that ⬎5% of genes require ⫹1 frameshifting for their expression and although this value is based upon only the
available 67 sequences, it suggests that programmed
frameshifting is very frequent in Euplotes. So far, six genes
have been confirmed to use ⫹1 frameshifting and of
these, four are involved in telomerase activity. The p43
protein identified in E. aediculatus is associated with
the active telomerase complex and probably facilitates
the binding of the catalytic subunit to the RNA. The ⫹1
frameshift is suspected to take place at an AAA UAA A
stretch by a mechanism dependent upon slow decoding
of the stop codon (UAA) in the ribosomal A site, allowing
tRNALys decoding the zero frame AAA codon in the P
site to slip ⫹1 onto the overlapping AAU codon and
subsequently, another lysyl-tRNA to decode the incoming ⫹1 frame codon (Aigner et al., 2000). In E. crassus,
three TERT genes encoding the telomerase catalytic
subunit have been identified. EcTERT2 expression requires a single ⫹1 frameshift event, probably at a AAA
UAA C stretch, whereas expression of EcTERT1 and 3
needs two sequential ⫹1 frameshifts, at the sequences
AAA UAA C and AAA UAA G (Karamysheva et al., 2003).
Similar to the telomerase-related examples, the other
two documented ⫹1 frameshift signals in Euplotes contain a “shifty” UAA stop codon (Weiss et al., 1987),
namely AAA UAA A (Tan et al., 2001a, 2001b). PKAR of E.
octocarinatus encodes a regulatory subunit of a cAMPdependent kinase and the EoNdr2 gene is a member of
the Ndr serine/threonine protein kinase family. In mechanistic terms, the presumed slow decoding of the UAA
stop codon may be a consequence of the use of UGA
as a cysteine codon. The changes within RF1 that allowed UGA to escape recognition (as a stop codon)
could well have compromised the ability of the release
factor to recognize UAA efficiently (Klobutcher and Farabaugh, 2002). Future work will determine if these ⫹1
frameshifting events have any regulatory function and
whether other mRNA elements are involved.
Two other examples of ⫹ 1 frameshifting in eukaryotes
warrant mention. The ABP140 gene of S. cerevisiae
(Asakura et al., 1998) encodes an actin binding protein
whose expression requires ⫹1 frameshifting at the slippery sequence CUU AGG C. This sequence is one of
the most underrepresented heptameric stretches in the
S. cerevisiae genome (11 found for 54 expected; Shah
et al., 2002) and promotes highly efficient frameshifting,
with about one in three ribosomes changing frame at
this site. It is closely related to that documented for
EST3 above and similarly, the potential involvement of
other mRNA elements needs to be addressed. Another
interesting example of ⫹1 programmed frameshifting
comes from the human IL-10 gene, encoding an immunosuppressive, antiinflammatory cytokine (Saulquin et
al., 2002). It has been shown that a cytotoxic T cell
epitope generated from the IL-10 gene by ⫹1 frameshifting could activate autoagressive T cells leading to the
elimination of the subset of cells producing this cytokine.
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161
However, that the epitope is generated by translational
frameshifting needs to be confirmed, as the proposed
slippery sequence is unconventional (CUU CCC U), with
no obvious flanking stimulatory elements.
Programmed -1 Frameshifting in Prokaryotes
Compared to ⫹1 frameshifting, few examples of –1 frameshifting in cellular genes have been described. Indeed, the
sole example with obvious biological relevance in prokaryotes is present in the dnaX gene, encoding the ␥ and ␶
subunits of DNA polymerase III holoenzyme. The holoenzyme is composed of three components; a DNA polymerase, a processivity factor (␤ sliding clamp), and a
clamp loader. Their association is required to obtain
high processivity during genomic DNA replication. In E.
coli, the ␶ subunit coordinates the synthesis of the leading and lagging strands by dimerization of the core polymerase. In addition, it mediates rapid replication fork
movement by its interaction with the DnaB helicase, and
affects the processivity during lagging strand synthesis
(Figure 4A). The ␥ subunits present in the clamp loader
play a crucial role in driving the ATP-induced conformational changes to the other subunits resulting in the
opening of the sliding clamp that then binds primed
DNA with high affinity. The synthesis of the ␥ protein
results from a -1 frameshifting event that directs ribosomes to a premature stop codon while the longest
form (␶) is translated by continued standard decoding
(Blinkowa and Walker, 1990; Flower and McHenry,
1990). The last 213 amino acids of the ␶ protein, missing
from the ␥ protein, confer the ability to bind directly the
core polymerase and DNA. The frameshift occurs on
the slippery sequence A AAA AAG, by simultaneous
slippage of both P and A site tRNALys species from the
zero (A AAA AAG) to the –1 frame (AAA AAA) (Jacks et
al., 1988; Tsuchihashi and Brown, 1992). This process
requires two stimulatory signals in the mRNA, an SDlike sequence 10 nt upstream of the slippery sequence
and a stem loop structure 5 nt downstream of it (Larsen
et al., 1994; see Figure 4B). In contrast to the RF2 signal
discussed earlier, the distance between this SD-like sequence and the start of the dnaX slippery sequence is
longer than that typically seen between the SD and the
AUG during initiation of translation and this likely leads
to some tension in the rRNA, promoting realignment in
the 5⬘ direction (pull-back). Furthermore, the A site slip
from AAG to AAA is favored by the stronger interaction
of tRNALys with the –1 frame AAA. Thus multiple features
are involved in this frameshift strategy. The conservation
of the frameshift signal in many Enterobacteriacea, the
related bacterium Vibrio cholerae, and in the more distant Neisseria genus might indicate an important function in the regulation of the ratio between the subunits
␶ and ␥ (Baranov et al., 2002). Remarkably, Thermus
thermophilus uses a transcriptional slippage mechanism rather than frameshifting to produce ␶ and ␥, although the ratio of the two proteins probably remains
the same (Larsen et al., 2000). Not all bacteria express
a ␥ subunit however, with dnaX encoding solely the ␶
subunit. The absence of ␥ is believed to generate a
polymerase whose action is less dedicated to the replication fork (Bruck et al., 2002).
An unusual –1 frameshift signal is present in the Bacil-
lus subtilis cytidine deaminase (CDA) gene (cdd) (Mejlhede et al., 1999; Licznar et al., 2003). CDAs are zinccontaining enzymes involved in the pyrimidine salvage
pathway and catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. The physiological relevance of the cdd frameshift
event is uncertain, since the C-terminal extension has
no apparent effect on CDA activity. The mechanism of
the frameshift however, is very unusual, involving –1
slippage of a single A site tRNALys at the slippery sequence CGA AAG (from AAG to AAA) but with the P site
tRNAArg appearing to remain bound in the zero phase,
with the wobble base of its anticodon being displaced
by the incoming lysyl-tRNA. An SD-like sequence 14 nt
upstream of the CGA AAG motif stimulates frameshifting
in a manner similar to that seen with dnaX. It has been
speculated that the cdd frameshift allows translational
regulation of the following gene, bex, (encoding a homolog of the E. coli membrane-bound G protein), since the
3⬘ end of cdd overlaps the 5⬘ end of bex. The SD-like
sequence present upstream of the cdd slippery sequence may be used to initiate translation at the bex
gene (Figure 5C). Under these conditions, a ribosomal
pause at the SD-like sequence during translation of the
cdd gene could prevent initiation at the bex gene (Mejlhede et al., 1999).
Two further examples of –1 frameshifting in prokaryotic cellular genes have been described, in the HpfucT2
gene of certain Helicobacter pylori strains (Wang et al.,
1999) and the fucA1 gene of Sulfolobus solfataricus (Cobucci-Ponzaro et al., 2003). To date, neither signal has
been characterized regarding the role of potential cisacting stimulatory elements and the frameshift events
remain to be confirmed in vivo.
Programmed -1 Frameshifting in Eukaryotes
Although many eukaryotic RNA viruses employ –1
frameshifting, there is only one example to date in a
conventional cellular gene, mouse Edr (embryonal carcinoma differentiation regulated), and this signal appears
to be a relic of retroviral origin (Shigemoto et al., 2001).
The frameshift signal of Edr is present between two long
overlapping ORFs and resembles a typical retrovirus
frameshift signal (Brierley and Pennell, 2001), with a slippery sequence G GGA AAC and a potential pseudoknotforming region five bases downstream. Pausing at the
pseudoknot is believed to promote simultaneous –1 slippage of both ribosome-bound tRNAs in manner similar
to that described for the dnaX frameshift signal. The
resemblance to viruses is increased when the coding
potential of the overlapping ORFs is taken into account.
ORF1 contains a putative zinc binding domain of the
CCHC subclass with a high content of basic amino acids
commonly found in retroviral Gag proteins, whereas the
protein encoded by the downstream ORF contains a
consensus motif for an aspartyl protease catalytic site.
Thus the organization is reminiscent of a retroviral gag/
pro overlap. The function of the gene is unknown but
the conservation of the frameshifting site in mouse and
human and the expression pattern during development
and in adult tissues argues for an important role for
frameshifting. Indeed, a more widespread involvement
of –1 frameshifting in eukaryotic gene expression seems
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Figure 4. Examples of –1 Frameshifting in Prokaryotic Genes
(A and B) Frameshifting in the E. coli dnaX gene. (A) Shows the structural organization of DNA polymerase III during the synthesis of the
leading and lagging strands. The ␶ (normal translation) and ␥ subunits (–1 frameshifting) are derived from the dnaX gene. (B) Organization of
the dnaX frameshift signal. Frameshifting occurs at the slippery sequence A AAA AAG (blue), enhanced by an upstream SD-like element
(purple) and a downstream stem-loop structure. The stop codon of the ␥ subunit, in the –1 frame, is shown in red. Panel A of Figure 4 was
reprinted from Leu, F., Georgescu, R. and O’Donnell, M. (2003). Mechanism of E. coli ␶ processivity. Switch during lagging strand synthesis.
Molecular Cell 11, 315-327. Copyright (2003) with permission from Elsevier.
(C) Ribosomal frameshifting in the B. subtilis cdd gene. Frameshifting occurs at the slippery sequence CGA AAG by an unusual single-tRNALys
slippage event in the ribosomal A-site, from AAG to the overlapping AAA codon in the –1 frame. An SD-like sequence (purple) can stimulate
the frameshift process or translation initiation at the start codon of the overlapping bex gene (orange). The cdd stop codon is indicated in red.
likely as judged from computer-assisted database screens
(Hammell et al., 1999).
Hopping
Hopping or translational bypassing during elongation
appears to be a rare event in natural mRNAs. The only
well-studied example is in gene 60 of bacteriophage T4,
where complex cis- and trans-acting signals allow a
50 nt stretch of mRNA to be bypassed at high efficiency
(Herr et al., 2000). There are no unquestionable examples
of bypassing in cellular genes, but the expression of the
Prevotella loescheii adhesin gene appears to require
bypassing of a 29 nt coding gap in the generation of
the SO34 protein (Manch-Citron et al., 1999). As yet, the
elements that promote the ribosomal gymnastics have
not been identified.
Recoding during Termination
Translation termination is an efficient process, essential
for the correct expression of proteins. The process is
achieved by release factors, three in prokaryotes (RF1,
RF2, RF3), and two in eukaryotes (eRF1 and eRF3). When
a stop codon is presented in the ribosomal A site, competition occurs between natural suppressor tRNAs and
class 1 release factors (RF1, RF2, eRF1) that bind the
stop codon and trigger release of the polypeptide chain.
RF1 recognizes UAA and UAG codons; RF2, UGA, and
UAA, whereas eRF1 recognizes all three stop codons.
Termination efficiency can be influenced by a number
of factors, including the nucleotide context of the stop
codon, particularly the base immediately 3⬘ of the stop,
the identity of the last two amino acids incorporated into
the polypeptide chain, and the presence of stimulatory
elements downstream (Bertram et al., 2001). These ele-
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163
Figure 5. Incorporation of Unusual Amino Acids at Stop Codons
(A) Insertion of selenocysteine at UGA codons requires a selenocysteine insertion element (SECIS). In eubacteria, a specialized translational
elongation factor SelB binds both the bSECIS element, located immediately downstream of the recoded UGA codon (in red), and the
selenocysteine-incorporating tRNA(Ser)Sec. In eukaryotes, the SECIS is located in the 3-untranslated region. Association of the eukaryotic SelB
ortholog mSelB (also known as eEFsec) requires an adaptor protein SBP2, the SECIS binding factor.
(B) Provides examples of SECIS elements from eubacteria (E. coli fdnG) and eukaryotes (H. sapiens selZ). Unconventional base pairs within
the selZ SECIS known to be essential for function are indicated in purple and the recoded UGA in red. Alongside the SECIS elements are
putative PYLIS elements (pyrrolysine insertion elements) located downstream of the recoded UAG codons (in red) of the mtmB1 and B2 genes of
various Methanosarcini strains. As mfold-derived structures (http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi), unconventional
base pairs of the kind seen in SECIS elements would not be identified.
ments can greatly increase the probability that a stop
codon will be decoded by a tRNA rather than a release
factor and so allow ribosomes to synthesize an elongated protein with potentially different biochemical properties. The most common programmed readthrough
event corresponds to the insertion of the amino acid
selenocysteine at UGA codons in both prokaryotes and
eukaryotes. In others cases, the flanking regions direct
the incorporation of a natural amino acid.
Incorporation of Unusual Amino Acids: the 21st
and 22nd Amino Acids
Selenium is an essential micronutrient for many organisms, including humans. The major biological form of
selenium is selenocysteine, which is obtained by biosynthesis from serine on a special tRNA, tRNA[Ser]Sec (Hatfield
and Gladyshev, 2002). In prokaryotes, the incorporation
of selenocysteine at a specific UGA site depends upon
tRNA[Ser]SecUCA, a specialized translation elongation factor
Molecular Cell
164
SelB and a structured RNA, bSECIS, a bacterial selenocysteine insertion sequence located immediately after the
UGA (Figure 5). In eukaryotes, perhaps as an evolutionary advantage gained from the uncoupling of transcription and translation, the SECIS elements are located in
the 3⬘UTR of the mRNA. One or two SECIS elements
are present and remarkably these can direct the incorporation of selenocysteine at multiple UGA codons within
the same mRNA. In the zebrafish Danio rerio, for example, expression of selenoprotein P requires the reassignment of 17 UGA codons, indicating that selenocysteine
insertion ought to be highly efficient (Tujebajeva et al.,
2000). However, the precise efficiency of selenocysteine
incorporation at various sites is an area of debate (Hatfield and Gladyshev, 2002; Krol, 2002; Driscoll and
Copeland, 2003). Some signals are genuinely inefficient
(eg: E. coli fdfF, 4%–5%, Suppmann et al., 1999) and as
premature termination is the consequence of failure to
insert selenocysteine, the cell must bear certain metabolic costs in order to accommodate this amino acid
into proteins. In prokaryotes, selenoproteins are primarily involved in catabolic processes and use selenium to
catalyze redox reactions, whereas in eukaryotes, selenoproteins participate in antioxidant and anabolic processes (reviewed in Hatfield and Gladyshev, 2002).
Although within the eukaryotic kingdom, sequenced
yeasts and higher plants do not appear to possess the
machinery for inserting selenocysteine into proteins, selenoproteins are essential for mammalian development,
as evidenced by the embryonic lethality observed in
knockout mice lacking tRNA[Ser]Sec (Bosl et al., 1997).
The UAG codon can trigger incorporation of pyrrolysine, the 22nd amino acid, which has recently been identified and found naturally in monomethylamine methyltransferases in Methanosarcina barkeri (Hao et al., 2002;
Srinivasan et al., 2002). This archaebacterium is a member of the methanogen group able to thrive on a wide
range of methanogenic substrates, including mono-, di-,
and trimethylamines. Each substrate requires activation
by a methyltransferase to generate methane. All known
methanogen methylamine methyltransferase genes contain an in frame UAG stop codon that we now know to
be read as pyrrolysine. The readthrough efficiency of
the UAG present in the mtmB1 gene of M. barkeri has
been estimated at more than 97%. The presence in this
organism of a tRNAPyl with anticodon 3⬘AUC5⬘ and studies
on the activation of this tRNA (Srinivasan et al., 2002;
Polycarpo et al., 2003) argue for cotranslational incorporation of pyrrolysine, but experimental support is lacking; for example, mRNA signals to subvert the meaning
of this particular UAG codon such as a PYLIS (pyrrolysine insertion sequence) element. However, putative
secondary structures with conserved features can be
predicted 5 or 6 nt downstream of the appropriate termination codon in the mtmB genes of three Methanosarcina strains (Figure 5). Whether these elements are required for pyrrolysine insertion requires investigation.
Stop Codon Readthrough in Prokaryotes
Contrary to popular belief, authentic examples of readthrough in prokaryotic genes are very scarce. The synthesis of the pathologically-important, adherent CS3 pilus of enterotoxigenic E. coli CFA/II strains requires the
expression of a 104 kDa protein (p104) by readthrough
and the presence of a suppressor tRNAGln is necessary
for efficient readthrough (Jalajakumari et al., 1989). In
the absence of the suppressor tRNA, p104 is still produced but at insufficient levels to support pilus formation. Another potential example of prokaryotic readthrough comes from the topo-3 gene of Bacillus firmus
OF4, encoding a protein with similarities to E. coli DNA
topoisomerase III (Ivey et al., 1992). This gene is interrupted by an in frame UGA, but suppression has not
been experimentally demonstrated. In fact, the stop codon is not conserved among other species, and at present this readthrough event must be viewed as speculative.
Stop Codon Readthrough in Eukaryotes
Recent studies of Drosophila developmental pathways
have revealed the occurrence of readthrough in three
genes, out at first (oaf) (Bergstrom et al., 1995), kelch
(Robinson and Cooley, 1997), and headcase (hdc) (Steneberg et al., 1998). The proteins encoded are involved
in diverse developmental processes. Oaf is expressed
during oogenesis in nurse cells and is widely distributed
throughout embryo development. It is found in embryonic, larval, and adult gonads of both sexes and its
inactivation can cause early death during larval development. Similarly, kelch is necessary for the production
of viable eggs in Drosophila ovaries; it is a structural
component of the ring canals that provide the intercellular conduits through which cytoplasm is transported
from the nurse cells to the oocyte in the egg chamber.
Headcase is involved in the regulation of tracheal
branching; it is expressed in a subset of tracheal fusion
cells and inhibits, in a dose-dependent manner, the terminal branching of neighboring cells. The signals that
promote readthrough in the three genes are uncertain
and the amino acid(s) inserted at the readthrough sites
have not been determined. The mechanism of readthrough is of great interest however, since in at least
two of the cases, the efficiency of readthrough appears
to vary during development. In ovarian cells, the oaf
readthrough product is minor, but in larvae and pupae,
it is expressed to levels similar to the non-readthrough
protein. Similarly, a spatial and temporal regulation of
readthrough in kelch has been observed, with the ratio
of the shorter and longer forms of kelch approaching
1:1 during metamorphosis, with the most efficient suppression in the imaginal discs and the testis. In oaf and
kelch, UGA is suppressed, raising the possibility that
selenocysteine insertion could be occurring, perhaps
supported by the observation that it was not possible
to detect the kelch readthrough product when the UGA
codon was replaced by UAA. However, the 3⬘ UTRs of
the two genes do not possess an obvious SECIS element, and at least in the case of kelch, it was not possible
to show incorporation of 75Se into the protein (Robinson
and Cooley, 1997). In hdc, the stop codon being suppressed is unusually UAA, commonly associated with
the highest termination efficiencies, yet the readthrough
is highly efficient, with about 20% of ribosomes continuing translation (Steneberg and Samakovlis, 2001). Computer-assisted secondary structure analysis predicts
the formation of stem-loop structures downstream of
Recoding in Cellular Gene Expression
165
the oaf, kelch, and hdc termination codons, especially
stable in the case of hdc, and these may play a role in
stimulating readthrough. In kelch, it is noticeable that
the triplets flanking the UGA are identical (AUG) and a
ribosomal hop over the stop codon could be taking
place rather than readthrough. This may simply be a
coincidence, but flanking sequences CCC AUG UGA
AUG GAA are conserved across Drosophila species.
Clearly, more work is needed to elucidate the molecular details.
Readthrough has also been described in the yeast
PDE2 gene, encoding a phosphodiesterase known to
bind to and degrade cAMP with high affinity. Readthrough extends the Pde2 polypeptide with a motif that
targets it for 26S proteasome degradation (Namy et al.,
2002). The efficiency of the suppression event is influenced by the environment; under normal circumstances
it is low (2%) but is enhanced about 10-fold in the absence of glucose and the presence of the yeast [PSI⫹]
factor. The degradation of a proportion of translated
Pde2 leads to an increase in the intracellular concentration of cAMP and a measured consequence of this is
reduced thermoresistance (Namy et al., 2002). As cAMP
is a major secondary messenger that controls, in response to environmental modifications, several biological processes including filamentation, stress response,
adaptation to carbon source, and cell cycle progression,
it seems likely that PDE2 readthrough will have a plethora of effects on yeast physiology. For example, it could
account for some of the numerous phenotypes associated with the appearance of the yeast prion [PSI⫹]
(Eaglestone et al., 1999; True and Lindquist, 2000).
Conclusions
Translational recoding events are associated with a variety of biological processes spanning the breadth of cellular expression strategies, from genomic replication
and maintenance through protein synthesis and degradation. In some cases, recoding is an autoregulatory
process, a tool for controlling gene expression in response to environmental factors. This is potentially of
evolutionary benefit; the regulation of recoding could
unveil silent genetic information by releasing the selective pressure on sequences downstream of the recoding
site, allowing it to evolve quickly. Subsequent stimulation of recoding by environmental factors would allow
the expression of proteins with potentially novel biochemical properties. This reversible adaptive process
could allow organisms to occupy a new ecological niche
without losing their capacity to occupy the old one,
as has been described for [PSI⫹] in yeast (True and
Lindquist, 2000) and Hsp90 in Arabidopsis (Queitsch et
al., 2002).
Recoding is clearly not limited to a subset of organisms, so one would envisage the identification, by genomic and proteomic studies, of numerous novel recoded
cellular genes within the next few years. However, the
prediction of recoding sites from genomic databases is
currently a difficult task. Two independent bioinformatics strategies are being developed to facilitate the identification of recoded genes in whole genomes. In modelbased approaches, genomic sequences are searched
for regions exhibiting a well-characterized recoding sig-
nal. Such analyses have already allowed the identification of several candidate recoded genes (Hammel et al.,
1999; Namy et al., 2002). The difficulty here is specificity,
with an imprecise model leading to the identification of
false positive candidates and too rigid a model failing
to identify truly positive candidates. Further, the identification of combinations of motifs involving primary
and secondary structural elements in the RNA is best
approached using stochastic context-free grammars
(SCFGs), but few practical SCGF programs are available
and are computationally complex, although efforts to reduce computational memory requirements are in progress
(Eddy, 2002). Of course, the parameters of the model
critically influence the outcome. For example, a model
based on our current knowledge of a viral recoding signal would probably miss distinct cellular counterparts.
For this reason, several groups are trying to develop
bioinformatics approaches that do not rely on models
of recoding sites and can be performed without a priori
knowledge of the mechanism involved. These similarity
based approaches seek genomic configurations compatible with recoding, such as ORFs separated by a
unique stop codon or overlapping along an intermediate
shared region. Several candidate recoded genes have
already been identified in yeast (Harrison et al., 2002;
Namy, et al., 2003) and in Drosophila (Sato et al., 2003)
in this way. The main difficulty is to discriminate between
active recoding sites, which play a physiological role,
and pseudogenes, undetected introns, or sequencing
errors. Naturally, the presence of known elements (stimulatory RNA structural motifs, SD-like sequences, nucleotide contexts) simplifies the identification of sites that
are likely to have biological significance. In the case of
selenoproteins, for example, the systematic presence
of the SECIS element has been used to identify 25 novel
selenoprotein genes in the human genome (Kryukov et
al., 2003). Similarly, comparative genome sequence analysis should be of benefit in the identification of bona
fide candidates.
There are clearly similarities between cellular recoding
events and those that take place in viruses and transposable elements, hinting at evolutionary links. For example, the –1 frameshift signals dnaX and Edr have
counterparts in prokaryotic insertional elements and
mammalian retroviruses, respectively (Baranov et al.,
2002); the ⫹1 frameshifting signals of the Euplotes TERT
genes are closely related to that used by the yeast retrotransposon Ty1 (Stahl et al., 2001), and the stop codon
readthrough signal of Drosophila hdc bears similarity to
that of the Dictostelium retrotransposon skipper (Bertram et al., 2001). Whether any of the cellular recoding
signals have been acquired from transposable elements
and viruses is unknown, but it is a possibility, especially
in the case of Edr (Shigemoto et al., 2001). There is,
paradoxically, good evidence of transmission the other
way, namely the acquisition of a human glutathione peroxidase selenoprotein homolog by the poxvirus Molluscum contagiosum (Shisler et al., 1998). Whatever their
evolutionary origins, a common feature of almost all
recoding signals appears to be a requirement for a
ribosomal pause, brought about by the use of slowly
decoded sense codons, termination codons, mRNA
secondary structures, or mRNA-rRNA interactions. In
mechanistic terms, pausing would increase the time at
Molecular Cell
166
which ribosomes are held at a recoding site, promoting
alternative events that would normally be unfavorable
kinetically. Although the individual elements that comprise a recoding signal undoubtedly have functions beyond simple induction of a pause, pausing is probably
a mechanistic requirement at most signals.
Over the next few years, it will be interesting to see
whether new recoding mechanisms will emerge. One
possibility is ribosomal skipping, described in the picornavirus foot and mouth disease virus. Here a termination
event occurs at a sense codon (without release factor
involvement), followed by release of the nascent polypeptide prior to ribosomal translocation to the next inframe codon (Donnelly et al., 2001). While the mechanism needs further analysis, the process seems to be
influenced by local sequences and could be used to
regulate the ratio between the synthesized polypeptides. Another possible recoding event would be the
regulation of amino acid misincorporation at sense codons leading to the modification of the activity, localization, or stability of the protein.
Recoding, especially readthrough, has gained biomedical attention recently in the use of aminoglycosides
to suppress disease-causing nonsense mutations (Howard et al., 2000; Sleat et al., 2001; Keeling and Bedwell,
2002). Although a promising new therapy for a large
number of genetic diseases, the drugs lack specificity at
present and their efficacy is influenced by the nucleotide
sequence identity flanking the termination codon in
question. A better understanding of the readthrough
mechanism coupled with specific drug design should
circumvent these problems allowing the use of this alternative treatment in numerous genetic diseases and
cancers.
Acknowledgments
We would like to express our gratitude to Alain Krol and Guillaume
Stahl for helpful discussions and to the maintainers of and contributors to the RECODE database (http://recode.genetics.utah.edu/cgibin/WebObjects/RecodeWeb.woa).
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