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The gene of an archaeal a-L-fucosidase is expressed
by translational frameshifting
Beatrice Cobucci-Ponzano
1
, Fiorella Conte
1
, Dario Benelli
2
, Paola Londei
2
,
Angela Flagiello
3
, Maria Monti
3
, Piero Pucci
3
, Mose
`Rossi
1,4
and Marco Moracci
1,
*
1
Institute of Protein Biochemistry—Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples,
Italy,
2
Dipartimento di Biochimica Medica e Biologia Medica (DIBIM), Universita
`di Bari—Piazzale Giulio Cesare,
70124 Bari, Italy,
3
CEINGE Biotecnologie Avanzate s.c.a r.l., Dipartimento di Chimica Organica e Biochimica,
Universita
`di Napoli Federico II, Complesso Universitario di Monte S. Angelo, via Cinthia 4, 80126 Napoli, Italy and
4
Dipartimento di Biologia Strutturale e Funzionale, Universita
`di Napoli ‘Federico II’, Complesso Universitario di Monte
S. Angelo, Via Cinthia 4, 80126 Naples, Italy
Received April 19, 2006; Revised July 21, 2006; Accepted July 22, 2006
ABSTRACT
The standard rules of genetic translational decoding
are altered in specific genes by different events
that are globally termed recoding. In Archaea reco-
ding has been unequivocally determined so far
only for termination codon readthrough events. We
study here the mechanism of expression of a gene
encoding for a a-L-fucosidase from the archaeon
Sulfolobus solfataricus (fucA1), which is split in two
open reading frames separated by a 1 frame-
shifting. The expression in Escherichia coli of
the wild-type split gene led to the production by
frameshifting of full-length polypeptides with an
efficiency of 5%. Mutations in the regulatory site
where the shift takes place demonstrate that the
expression in vivo occurs in a programmed way.
Further, we identify a full-length product of fucA1
in S.solfataricus extracts, which translate this gene
in vitro by following programmed 1 frameshifting.
This is the first experimental demonstration that
this kind of recoding is present in Archaea.
INTRODUCTION
Translation is optimally accurate and the correspondence
between the nucleotide and the protein sequences are often
considered as an immutable dogma. However, the genetic
code is not quite universal: in certain organelles and in a
small number of organisms the meaning of different codons
has been reassigned and all the mRNAs are decoded
accordingly. More surprisingly, the standard rules of genetic
decoding are altered in specific genes by different events that
are globally termed recoding (1). In all cases, translational
recoding occurs in competition with normal decoding, with
a proportion of the ribosomes not obeying to the ‘universal’
rules. Translational recoding has been identified in both
prokaryotes and eukaryotes. It has crucial roles in the regula-
tion of gene expression and includes stop codon readthrough,
ribosome hopping and ±1 programmed frameshifting [for
reviews see (2–4)].
In stop codon readthrough a stop codon is decoded by a
tRNA carrying an unusual amino acid rather than a transla-
tional release factor. Specific stimulatory elements down-
stream to the stop codon regulate this process (5). Hopping,
in which the ribosome stops translation in a particular site
of the mRNA and re-start few nucleotides downstream, is a
rare event and it has been studied in detail only in the bacte-
riophage T4 (6). In programmed frameshifting, ribosomes are
induced to shift to an alternative, overlapping reading frame
1nt3
0-wards (+1 frameshifting) or 50-wards (1 frameshifting)
of the mRNA. This process is regulated and its frequency
varies in different genes. The ±1 programmed frameshifting
has been studied extensively in viruses, retrotransposons and
insertion elements for which many cases are documented
(7–9). Instead, this phenomenon is by far less common in
cellular genes. A single case of programmed +1 frameshift-
ing is known in prokaryotes (10,11) while in eukaryotes,
including humans, several genes regulated by this recoding
event have been described previously [(4) and references
therein]. Compared to +1 frameshifting, 1 frameshifting
is less widespread with only two examples in prokaryotes
(12–14) and few others in eukaryotes (15–17).
The programmed 1 frameshifting is triggered by several
elements in the mRNA. The slippery sequence, showing the
X-XXY-YYZ motif, in which X can be any base, Y is usually
A or U, and Z is any base but G, has the function of favouring
the tRNA misalignment and it is the site where the shift takes
place (3,18). Frameshifting could be further stimulated by
other elements flanking the slippery sequence: a codon for
a low-abundance tRNA, a stop codon, a Shine–Dalgarno
sequence and an mRNA secondary structure. It has been
*To whom correspondence should be addressed. Tel: +39 081 6132271; Fax: +39 081 6132277; Email: m.moracci@ibp.cnr.it
2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
4258–4268 Nucleic Acids Research, 2006, Vol. 34, No. 15 Published online 18 August 2006
doi:10.1093/nar/gkl574
reported that these elements, alone or in combination,
enhance frameshifting by pausing the translating ribosome
on the slippery sequence (4,18).
Noticeably, known cases of recoding in Archaea [recently
reviewed in (19)] are limited to termination codon
readthrough events that regulate the incorporation of the
21st and 22nd amino acids selenocysteine and pyrrolysine,
respectively (20–23).
No archaeal genes regulated by translational programmed
frameshifting and ribosome hopping have been identified
experimentally so far; therefore, if compared with the others
domains of life, the study of translational recoding in Archaea
is still at its dawn.
We showed that the a-L-fucosidase gene from the crenar-
chaeon Sulfolobus solfataricus is putatively expressed by pro-
grammed 1 frameshifting (24). This gene, named fucA1,is
organized in the open reading frames (ORFs) SSO11867 and
SSO3060 of 81 and 426 amino acids, respectively, which are
separated by a 1 frameshifting in a 40 base overlap
(Figure 1A). We have reported previously that the region of
overlap between the two ORFs had the characteristic features
of the genes expressed by programmed 1 frameshifting
including a slippery heptanucleotide A-AAA-AAT (codons
are shown in the zero frame) flanked by a putative stem–
loop and the rare codons CAC (Figure 1A) resembling the
prokaryotic stem–loops/hairpins and the Shine–Dalgarno-
like sites (24). We showed that the frameshifting, obtained
by mutating by site-directed mutagenesis the fucA1 gene
exactly in the position predicted from the slippery site,
produced a full-length gene, named fucA1
A
, encoding for a
polypeptide of 495 amino acids (Figure 1B). This mutant
gene expressed in Escherichia coli a fully functional
a-L-fucosidase, named Ssa-fuc, which was thermophilic,
thermostable and had an unusual nonameric structure
(24,25). More recently, we determined the reaction mecha-
nism and the function of the residues of the active site of
the mutant enzyme (26,27).
The functionality of the product of the mutant gene
fucA1
A
does not provide direct experimental evidence that
programmed 1 frameshifting occurs in vivo and in
S.solfataricus. To address these issues, we report here the
study of the expression of the wild-type split gene fucA1
and of its mutants in the slippery sequence. We demonstrate
here that fucA1 is expressed by programmed 1 frameshift-
ing in both E.coli and S.solfataricus. This is the first experi-
mental demonstration that this kind of recoding is present in
the Archaea domain of life. The relevance of programmed 1
frameshifting in Archaea is also discussed.
Figure 1. The a-fucosidase gene. (A) Region of overlap in the wild-type split fucA1 gene. The N-terminal SSO11867 ORF is in the zero frame, the C-terminal
SSO3060 ORF, for which only a fragment is shown, is in the 1 frame. The slippery heptameric sequence is underlined; the rare codons are boxed and the arrows
indicate the stems of the putative mRNA secondary structure. The amino acids involved in the programmed 1 frameshifting and the first codon translated after
this event in the 1 frame are shadowed. (B) Fragment of the full-length mutant fucA1
A
gene. The small arrows indicate the mutated nucleotides.
Nucleic Acids Research, 2006, Vol. 34, No. 15 4259
MATERIALS AND METHODS
Analysis of the a-fucosidase expression
S.solfataricus cells were grown, and cell extracts obtained, as
described previously (24,28).
The expression in the E.coli strain BL21(RB791) of
the wild-type gene fucA1 and of the mutant genes fucA1
A
[previously named FrameFuc in (24)], fucA1
B
,fucA1
sm
and
fucA1
tm
as fusions of glutathione S-transferase (GST) and
the purification of the recombinant proteins were performed
as reported previously (23). The nomenclature used in this
paper for the different a-fucosidase genes is listed in Table 1.
For the western blot studies, equal amounts of E.coli
cultures expressing the wild-type and mutant fucA1 genes,
normalized for the OD
600
, were resuspended in SDS–PAGE
loading buffer containing 0.03 M Tris–HCl buffer, pH 6.8,
3% SDS (w/v), 6.7% glycerol (w/v), 6.7% 2-mercaptoethanol
(w/v) and 0.002% blue bromophenol (w/v). The samples were
incubated at 100C for 5 min (unless otherwise indicated)
and were directly loaded on to the gel. Western blot analyses
were performed by blotting SDS–PAGEs of the concen-
trations indicated on Hybond-P polyvinylidenfluorid filters
(Amersham Biosciences, Uppsala, Sweden); polyclonal
anti-Ssa-fuc antibodies from rabbit (PRIMM, Milan, Italy)
and anti-GST antibodies (Amersham Biosciences) were
diluted 1:5000 and 1:40 000, respectively. The filters were
washed and incubated with the ImmunoPure anti-rabbit IgG
antibody conjugated with the horseradish peroxidase (HRP)
from Pierce Biotechnology (Rockford, IL, USA). Filters
were developed with the ECL-plus Western Blotting Detec-
tion system (Amersham Biosciences) by following the manu-
facturer’s indications. The molecular weight markers used in
the western blot analyses were the ECL streptavidin–HRP
conjugate (Amersham Biosciences).
The protein concentration of the samples was measured
with the method of Bradford (29) and the amounts of
sample loaded on to the SDS–PAGEs are those indicated.
The quantification of the bands identified by western blot
was performed by using the program Quantity One 4.4.0 in a
ChemiDoc EQ System (Bio-Rad, Hercules, CA, USA) with
the volume analysis tool. The frameshifting efficiency was cal-
culated as the ratio of the intensity of the bands of the frame-
shifted product/frameshifted product +termination product.
The mutants in the slippery sequence of the wild-type gene
fucA1 were prepared by site-directed mutagenesis from the
vector pGEX-11867/3060, described previously (24,27).
The synthetic oligonucleotides used (PRIMM) were the
following: FucA1sm-rev, 50-TTTAGGTGATATTGGTGTT-
CTGGTCTATCT-30; FucA1sm-fwd, 50-GAACACCAATAT-
CACCTAAAGAATTCGGCCCA-30; FucA1tm-rev, 50-AGG-
TGATATTGGTGTTCTGGTCTATCTGGC-30; FucA1tm-fwd,
50-CCAGAACACCAATATCACCTCAAGAACTCGGCCCA-
GT-30, where the mismatched nucleotides in the mutagenic
primers are underlined. Direct sequencing identified the plas-
mids containing the desired mutations and the mutant genes,
named fucA1
sm
and fucA1
tm
, were completely re-sequenced.
Expression and characterization of Ssa-fuc
B
The mutant Ssa-fuc
B
was prepared by site-directed muta-
genesis from the vector pGEX-11867/3060, by using the
same site-directed mutagenesis kit described above. The syn-
thetic oligonucleotides used were FucA1sm-rev (described
above) and the following mutagenic oligonucleotide: Fuc-B,
50-GAACACCAATATCACCTAAAGAAGTTCGGCCC-
AGT-30, where the mismatched nucleotides are underlined.
Direct sequencing identified the plasmid containing the desired
mutations and the mutant gene, named fucA1
B
, was completely
re-sequenced. The enzymatic characterization of Ssa-fuc
B
was
performed as described previously (24,27).
Mass spectrometry experiments
Samples of the proteins expressed in E.coli from the wild-
type gene fucA1 and the mutants fucA1
A
and fucA1
sm
, purified
as described, were fractionated on an SDS–PAGE. Protein
bands were excised from the gel, washed in 50 mM ammo-
nium bicarbonate, pH 8.0, in 50% acetonitrile, reduced with
10 mM DTT at 56C for 45 min and alkylated with 55 mM
iodoacetamide for 30 min at room temperature in the dark.
The gel pieces were washed several times with the buffer,
resuspended in 50 mM ammonium bicarbonate and incubated
with 100 ng of trypsin for 2 h at 4C and overnight at
37C. The supernatant containing peptides was analysed by
MALDIMS on an Applied Biosystem Voyager DE-PRO
mass spectrometer using a-cyano-4-hydroxycynnamic acid
as matrix. Mass calibration was performed by using the
standard mixture provided by manufacturer.
Liquid chromatography online tandem mass spectrometry
(LCMSMS) analyses were performed on a Q-TOF hybrid
mass spectrometer (Micromass, Waters, Milford, MA,
USA) coupled with a CapLC capillary chromatographic sys-
tem (Waters). Peptide ions were selected in the collision cell
and fragmented. Analysis of the daughter ion spectra led to
the reconstruction of peptide sequences.
Experiments of translation in vitro
Genomic DNA from S.solfataricus P2 strain was prepared as
described previously (24). A DNA fragment of 1538 nt con-
taining the complete fucA1 gene, was prepared by PCR, by
using the following synthetic oligonucleotides (Genenco,
Florence, Italy): FucA1-fwd, 50-CTGGAGGCGCGCTAA-
TACGACTCACTATAGGTCAGTTAAATGTCACAAAA-
TTCT-30; FucA1-rev, 50-GACTTGGCGCGCCTATCTAT-
AATCTAGGATAACCCTTAT-30, in which the sequence
corresponding to the genome of S.solfataricus is underlined.
In the FucA1-fwd primer, the sequence of the promoter of
the T7 RNA polymerase is in boldface and the sequence of
the BssHII site is shown in italics. The PCR amplification
was performed as described previously (24) and the ampli-
fication products were cloned in the BssHII site of the plas-
mid pBluescript II KS+.ThefucA1 gene was completely
re-sequenced to check if undesired mutations were introduced
by PCR and the recombinant vector obtained, named pBlu-
FucA1, was used for translation in vitro experiments.
The plasmids expressing the mutant genes fucA1
A
,fucA1
sm
and fucA1
tm
for experiments of translation in vitro were pre-
pared by substituting the KpnI–NcoI wild-type fragment,
containing the slippery site, with those isolated from the
mutants. To check that the resulting plasmids had the correct
sequence, the mutant genes were completely re-sequenced.
4260 Nucleic Acids Research, 2006, Vol. 34, No. 15
The mRNAs encoding wild-type fucA1 and its various
mutants were obtained by in vitro run-off transcription.
About 2 mg of each plasmid was linearized with BssHII
and incubated with 50 U of T7 RNA polymerase for 1 h
30 min at 37C. The transcription mixtures were then treated
with 10 U of DNAseI (RNAse free) for 30 min. The tran-
scribed RNAs were recovered by extracting the samples
twice with phenol (pH 4.7) and once with phenol/chloroform
1:1 followed by precipitation with ethanol. The mRNAs
were resuspended in DEPC-treated H
2
O at the approximate
concentration of 0.6 pmol/ml.
In vitro translation assays were performed essentially as
described by Condo
`et al. (28). The samples (25 ml final
volume) contained 5 mlofS.solfataricus cell extract,
10 mM KCl, 20 mM Tris–HCl, pH 7.0, 20 mM Mg acetate,
3 mM ATP, 1 mM GTP, 5 mg of bulk S.solfataricus tRNA,
2mlof[
35
S]methionine (1200 Ci/mmol at 10 mCi/ml) and
10 pmol of each mRNA. The mixtures were incubated at
70C for 45 min. After this time, the synthesized proteins
were resolved by electrophoresis 12.5% acrylamide–SDS
gels and revealed by autoradiography of the dried gels on
an Instant Imager apparatus.
Transcriptional analysis of fucA1
Cells of S.solfataricus, strain P2, were grown in minimal
salts culture media supplemented with yeast extract (0.1%),
casamino acids (0.1%), plus glucose (0.1%) (YGM) or
sucrose (0.1%) (YSM). The extraction of total RNA was
performed as reported previously (24). Total RNA was
extensively digested with DNAse (Ambion, Austin, TX,
USA) and the absence of DNA was assessed by the lack
of PCR amplification with each sets of primers described
below. The RT–PCR experiments were performed as
reported previously (24) by using the primers described pre-
viously that allowed the amplification of a region of 833 nt
(positions 1–833, in which the A of the first ATG codon is
numbered as one) overlapping the ORFs SSO11867 and
SSO3060 (24).
For real-time PCR experiments total cDNA was obtained
using the kit Quantitect RT (Qiagen GmbH, Hilden,
Germany) from 500 ng of the same preparation of RNA
described above. cDNA was then amplified in a Bio-Rad
LightCycler using the DyNAmo HS Syber Green qPCR Kit
(Finnzymes Oy, Espoo, Finland). Synthetic oligonucleotides
(PRIMM) used for the amplification of a region at the 30of
the ORF SSO3060 were as follows: 50-Real: 50-TAAATGGC-
GAAGCGATTTTC-30;3
0-Real: 50-ATATGCCTTTGTCGC-
GGATA-30for the gene fucA1.5
0-GAATGGGGGTGATA-
CTGTCG-30and 50-TTTACAGCCGGGACTACAGG-30for
the 16S rRNA gene.
For each amplification of the fucA1 gene was used 2500-
fold more cDNA than that used for the amplification of the
16S rRNA. Controls with no template cDNA were always
included. PCR conditions were 15 min at 95C for initial
denaturation, followed by 40 cycles of 10 s at 95C, 25 s at
56C and 35 s at 72C, and a final step of 10 min at 72C.
Product purity was controlled by melting point analysis of
setpoints with 0.5C temperature increase from 72 to 95C.
PCR products were analysed on 2% agarose gels and visual-
ized by ethidium bromide staining.
The expression values of fucA1 gene were normalized to
the values determined for the 16S rRNA gene. Absolute
expression levels were calculated as fucA1/16S ratio in
YSM and YGM cells, respectively. Relative mRNA expres-
sion levels (YSM/YGM ratio) were calculated as ( fucA1/
16S ratio in YGM cells)/(fucA1/16S ratio in YSM). Each
cDNA was used in triplicate for each amplification.
RESULTS
Expression of fucA1 in E.coli
The wild-type fucA1 gene, expressed in E.coli as a GST-fused
protein, produced trace amounts of a-fucosidase activity
(2.3 ·10
2
units mg
1
after removal of GST), suggesting
that a programmed 1 frameshifting may occur in E.coli
(24). The enzyme was then purified by using the GST puri-
fication system and analysed by SDS–PAGE revealing a
major protein band (Figure 2A). The sample and control
bands were excised from the gel, digested in situ with trypsin
and directly analysed by matrix-assisted laser desorption/
ionization mass spectrometry (MALDIMS). As shown in
Figure 2B and C, both spectra revealed the occurrence of
an identical mass signal at m/z 1244.6 corresponding to a
peptide (Peptide A) encompassing the overlapping region of
the two ORFs. This result was confirmed by liquid chro-
matography online tandem mass spectrometry (LCMSMS)
analysis of the peptide mixtures. The fragmentation spectra
of the two signals showed the common sequence Asn-Phe-
Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys in which the amino
acid from the ORF SSO11867 is underlined. These results
unequivocally demonstrate that the protein containing the
Peptide A is produced in E.coli by a frameshifting event
that occurred exactly within the slippery heptamer predicted
from the analysis of the DNA sequence in the region of over-
lap between the ORFs SSO11867 and SSO3060 (Figure 1A).
Remarkably, the MALDIMS analysis of the products of
the wild-type fucA1 gene revealed the presence of a second
Peptide B at m/z 1258.6 that is absent in the spectra of the
Ssa-fuc control protein (Figure 2B and C). The sequence of
Peptide B obtained by LCMSMS (Figure 2D) was Lys-Phe-
Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys. This sequence dif-
fers only by one amino acid from Peptide A demonstrating
that the interrupted gene fucA1 expresses in E.coli two full-
length proteins originated by different 1 frameshifting
events. Polypeptide A results from a shift in a site A and it
is identical to Ssa-fuc prepared by site-directed mutagenesis
(24), suggesting that the expression occurred with the simul-
taneous P- and A-site slippage. Instead, polypeptide B, named
Ssa-fuc
B
, is generated by frameshifting in a second site B as
the result of a single P-site slippage (Figure 2E).
To measure the global efficiency of frameshifting in the
two sites of the wild-type gene fucA1 we analysed the total
extracts of E.coli by western blot using anti-GST antibodies
(Figure 2F). Two bands with marked different electrophoretic
mobility were observed: the polypeptide of 78.7 ± 1.1 kDa
migrated like GST-Ssa-fuc fusion and was identified as origi-
nated from frameshifting in either site A or B of fucA1. The
protein of 38.1 ± 1.2 kDa, which is not expressed by the mut-
ant gene fucA1
A
(not shown), had an electrophoretic mobility
compatible with GST fused to the polypeptide encoded by the
Nucleic Acids Research, 2006, Vol. 34, No. 15 4261
ORF SSO11867 solely (27 and 9.6 kDa, respectively). This
polypeptide originated from the translational termination of
the ribosome at the OCH codon of the fucA1 N-terminal
ORF (Figure 1A). The calculated ratio of frameshifting to
the termination products was 5%.
Preparation and characterization of Ssa-fuc
B
To test if the full-length a-fucosidase produced by the 1
frameshifting event in site B (Ssa-fuc
B
), resulting from the
single P-site slippage has different properties from Ssa-fuc,
whose sequence arises from the simultaneous P- and A- site
slippage, we prepared the enzyme by site-directed muta-
genesis. The slippery sequence in fucA1 A-AAA-AAT was
mutated in A-AAG-AAG-T where mutations are underlined.
The new mutant gene was named fucA1
B
. The first G, produc-
ing the conservative mutation AAA!AAG, was made to
disrupt the slippery sequence and hence reducing the shifting
efficiency. The second G was inserted to produce the
frameshifting that results in the amino acid sequence of
Peptide B. Therefore, the sequence of the two full-length
mutant genes fucA1
A
and fucA1
B
differs only in the region
of the slippery sequence: A-AAG-AAT-TTC-GGC and
A-AAG-AAG-TTC-GGC, respectively (the mutations are
underlined, the nucleotides in boldface were originally in
the 1 frame) (Table 1).
The recombinant Ssa-fuc
B
was purified up to 95%
(Materials and Methods). Gel filtration chromatography demon-
strated that in native conditions Ssa-fuc
B
had the same non-
americ structure of Ssa-fuc with an identical molecular weight
of 508 kDa (data not shown). In addition, Ssa-fuc
B
had the same
high substrate selectivity of Ssa-fuc. The two enzymes have
high affinity for 4-nitrophenyl-a-L-fucoside (4NP-Fuc) sub-
strate at 65C; the K
M
is identical within the experimental
error (0.0287 ± 0.005 mM) while the k
cat
of Ssa-fuc
B
(137 ±
5.7 s
1
)is48%ofthatofSsa-fuc (287 ± 11 s
1
). In addition,
4-nitrophenyl-a-L-arabinoside, -rhamnoside, 4-nitrophenyl-a-
D-glucoside, -xyloside, -galactoside and -mannoside were not
substrates of Ssa-fuc
B
as shown previously for Ssa-fuc (24).
This suggests that the different amino acid sequence did not
Figure 2. Analysis of the expression of fucA1 in E.coli.(A) Coomassie stained 7% SDS–PAGE showing (line 1) the recombinant Ssa-fuc (3 mg) and (line 2) the
purified products of the wild-type split fucA1 gene. The protein concentration of the latter sample could not be quantified because of the scarcity of
the purification yields. MALDIMS of the purified products of the wild-type fucA1 gene and of Ssa-fuc are shown in (B) and (C), respectively. Peptide A and B
are indicated. (D) LCMSMS analysis of peptide B. (E) The proposed frameshifting sites in the fucA1 gene. The open and the closed arrows indicate the shifting
sites A and B, respectively (for details see text). The sequence of the peptides A and B are also indicated. (F) Western blot of E.coli cellular extracts expressing
the wild-type fucA1 gene. The blot was probed with anti-GST antibodies. The pre-stained molecular weight markers were b-galactosidase (175 000), paramyosin
(83 000), glutamic dehydrogenase (62 000), aldolase (47 500) and triosephosphate isomerase (32 500).
Table 1. Nomenclature and characteristics of the a-fucosidase genes
Gene name Status Name of the
recombinant protein
Slippery heptamer
a
fucA1 wild type 1 frameshifted — A-AAA-AAT
fucA1
A
mutant Full-length Ssa-fuc A-AAG-AAT
fucA1
B
mutant Full-length Ssa-fuc
B
A-AAG-AAG
fucA1
sm
mutant 1 frameshifted — A-AAG-AAT
fucA1
tm
mutant 1 frameshifted — C-AAG-AAC
a
Nucleotides modified by substitution and insertion mutations are underlined
and in boldface, respectively.
4262 Nucleic Acids Research, 2006, Vol. 34, No. 15
significantly affect the active site. Both enzymes showed an
identical profile of specific activity versus temperature with an
optimal temperature higher than 95C (data not shown). The
heat stability and the pH dependence of Ssa-fuc and Ssa-fuc
B
are reported in Figure 3. At 80C, the optimal growth
temperature of S.solfataricus, the half-life of Ssa-fuc
B
is
45 min, almost 4-fold lower than that of Ssa-fuc (Figure 3A).
The two enzymes showed different behaviour at pH <6.0 at
which Ssa-fuc
B
is only barely active and stable (Figure 3B);
however, the two enzymes showed similar values of specific
activity at pHs above 6.0, which is close to the intracellular pH
of S.solfataricus (30).
Characterization of the slippery sequence of
fucA1 in E.coli
The experimental data reported above indicate that the pre-
dicted slippery heptanucleotide in the region of overlap
between the ORFs SSO11867 and SSO3060 of the wild-
type gene fucA1 could regulate in cis the frameshifting events
observed in E.coli. To test this hypothesis, we mutated the
sequence A-AAA-AAT into A-AAG-AAT and C-AAG-
AAC (mutations are underlined) obtaining the fucA1 single
mutant ( fucA1
sm
) and triple mutant ( fucA1
tm
) genes, respec-
tively. It is worth noting that the mutations disrupt the
slippery sequence, but they maintain the 1 frameshift
between the two ORFs (Table 1).
Surprisingly, the expression of fucA1
sm
in E.coli produced
a full-length polypeptide that, after purification by affinity
chromatography and removal of the GST protein, showed
the same electrophoretic migration of Ssa-fuc and Ssa-fuc
B
(Figure 4A). This protein was then characterized by mass
spectrometry analyses following in situ tryptic digestion.
Interestingly, the MALDI spectra revealed the presence of a
single peptide encompassing the overlapping region between
the two ORFs with a mass value of 1259.7 Da (peptide C;
Figure 4B). The sequence of peptide C, determined from
the fragmentation spectra obtained by LCMSMS analysis, was
Glu-Phe-Gly-Pro-Val-Thr-Asp-Phe-Gly-Tyr-Lys (Figure 4C).
Remarkably, apart from the Glu residue, this sequence is
identical to that of peptide B produced from fucA1, indicat-
ing that in the mutant gene fucA1
sm
only one of the two
frameshifting events observed in the wild-type fucA1 gene
had occurred. The presence of a Glu instead of Lys was
not unexpected. The mutation A-AAA-AAT!A-AAG-
AAT in fucA1
sm
was conservative in the zero frame of the
ORF SSO11867 (AAA!AAG, both encoding Lys), but it
produced the mutation AAA!GAA (Lys!Glu) in the 1
frame of the ORF SSO3060.
It is worth noting that the frameshifting efficiency of the
gene fucA1
sm
, calculated by western blot as described
above, was 2-folds higher (10%) if compared to fucA1 (5%)
(Figure 4D). This indicates that the mutation cancelled the
frameshifting site A and, in the same time, enhanced the
frameshifting efficiency of site B.
In contrast, the triple mutant fucA1
tm
produced in E.coli
only the low molecular weight band resulting from transla-
tional termination (Figure 4D). No full-length protein could
be detected in western blots probed with either anti-GST
(Figure 4D) or anti-Ssa-fuc antibodies (Figure 4E). These
data show that the disruption of the heptameric slippery
sequence completely abolished the frameshifting in E.coli
confirming that this sequence has a direct role in controlling
the frameshifting in vivo.
Expression of fucA1 in S.solfataricus
To test whether fucA1 is expressed in S.solfataricus we anal-
ysed the extracts of cells grown on yeast extract, sucrose and
casaminoacids medium (YSM). Accurate assays showed that
S.solfataricus extracts contained 3.4 ·10
4
units mg
1
of
a-fucosidase activity. These very low amounts hampered
the purification of the enzyme. The extracts of S.solfataricus
cells grown on YSM revealed by western blot a band of a
molecular mass >97 kDa and no signals were detected with
the pre-immune serum confirming the specificity of the
anti-Ssa-fuc antibodies (Figure 5A). The different molecular
mass may result from post-translational modifications
occurred in the archaeon or from the incomplete denaturation
of a protein complex. In particular, the latter event is not
unusual among enzymes from hyperthermophilic archaea
(31,32). To test which hypotheses were appropriate, cellular
extracts of S.solfataricus were analysed by western blot
extending the incubation at 100C to 2 h. Interestingly, this
Figure 3. Comparison of the stability and pH dependence of Ssa-fuc and
Ssa-fuc
B
.(A) Thermal stability of Ssa-fuc (open circles) and Ssa-fuc
B
(closed circles) at 80C. (B) pH dependence of Ssa-fuc (open circles) and
Ssa-fuc
B
(closed circles) at 65C.
Nucleic Acids Research, 2006, Vol. 34, No. 15 4263
treatment shifted the high-molecular mass band to 67.6 ± 1.2
kDa (Figure 5B and C), which still differs from that of the
recombinant Ssa-fuc, 58.9 ± 1.2 kDa, leaving the question
on the origin of this difference unsolved. To try to shed
some light we immunoprecipitated extracts of S.solfataricus
with anti-Ssa-fuc antibodies and we analysed the major
protein band by MALDIMS. Unfortunately, we could not
observe any peptide compatible with the fucosidase because
the heavy IgG chain co-migrated with the band of the
expected molecular weight (data not shown).
To test if the scarce amounts of the a-fucosidase in
S.solfataricus extracts was the result of reduced expression
at transcriptional level, we performed a northern blot analysis
of total RNA extracted from cells grown either on YSM or
YGM media. We could not observe any signal by using
probes matching the 30of the ORF SSO3060 (data not
shown). These results suggest that fucA1 produced a rare tran-
script; therefore, we analysed the level of mRNA by RT–PCR
and by real-time PCR. A band corresponding to the region of
overlap between the ORFs SSO11867 and SSO3060 was
observed in the RNA extracted from cells grown on YSM
Figure 5. Analysis of the expression of the a-fucosidase in S.solfataricus.
(A) Western blot analysis of recombinant Ssa-fuc (lanes 1, 2, 5 and 6,
0.14 mg) and of extracts of S.solfataricus cells grown on YSM (lanes 3, 4, 7
and 8, 153 mg). Samples in lanes 1, 3, 5 and 7 were not denaturated
before loading. The left panel shows the blot probed with anti-Ssa-fuc
antibodies; the right panel was probed with the pre-immune serum diluted
1:5 000. (B) Western blot analysis: recombinant Ssa-fuc (lanes 1, 2 and 3,
0.5 mg) incubated at 100C for 5 min, 1 h and 2 h, respectively; extracts
of S.solfataricus cells (lanes 4, 5 and 6, 1 mg) incubated at 100C for 5 min,
1 h and 2 h, respectively. (C) Western blot analysis of recombinant
Ssa-fuc (lane 1, 0.1 mg) incubated at 100C for 5 min and of extracts of
S.solfataricus cells (lane 2, 1 mg) incubated at 100C for 2 h, respectively.
The molecular weight markers were: phosphorylase b (97 000), albumin
(66 000), ovalbumin (45 000), carbonic anhydrase (31 000) and trypsin
inhibitor (20 100).
Figure 4. Analysis of the expression in E.coli of the mutants in the slippery sequence. (A) Coomassie stained 7% SDS–PAGE showing (arrow) the purified
recombinant Ssa-fuc
B
(1.2 mg), the product of the gene fucA1
sm
(2 mg), and Ssa-fuc (4 mg). The bands with faster electrophoretic mobility result from the
proteolytic cleavage of the full-length protein (25). (B) Partial MALDIMS spectrum of the tryptic digest from mutant fucA1
sm
expressed in E.coli. The mass
signal corresponding to peptide C encompassing the overlapping region is indicated. (C) LCMSMS analysis of peptide C. The amino acid sequence inferred from
fragmentation spectra is reported. (D) Western blot of E.coli cellular extracts expressing fucA1
A
, the wild-type fucA1,fucA1
sm
and fucA1
tm
genes (Materials and
Methods). The blot was probed with anti-GST antibodies. (E) Western blot of partially purified protein samples expressed in E.coli fused to GST from wild-type
and mutant fucA1 genes. Cellular extracts were loaded on GST–Sepharose matrix. After washing, equal amounts of slurries (30 ml of 300 ml) were denaturated
and loaded on a 8% SDS–PAGE. Extracts of E.coli cells expressing the parental plasmid pGEX-2TK were used as the negative control (pGEX). The blot was
probed with anti-Ssa-fuc antibodies.
4264 Nucleic Acids Research, 2006, Vol. 34, No. 15
and YGM media, demonstrating that under these conditions
the two ORFs were co-transcribed (Figure 6A).
The experiments of real-time PCR shown in Figure 6B
demonstrated that rRNA16S was amplified after 17 cycles
while the amplification of fucA1 mRNA was observed after
38 cycles, despite the fact that we used 2500-fold more
cDNA for the amplification of fucA1. This indicates that
the gene fucA1 is transcribed at very low level. No significant
differences in the fucA1 mRNA level were observed in cells
grown in YSM or YGM media. This is further confirmed by
the analysis by western blot of the extracts of the same cells
of S.solfataricus used to prepare the total RNAs, which
revealed equal amounts of a-fucosidase in the two extracts
(Figure 6C). Therefore, the low a-fucosidase activity
observed under the conditions tested is the result of the
poor transcription of the fucA1 gene.
Analysis of the expression of fucA1 in S.solfataricus by
in vitro translation
To determine whether, and with what efficiency, the 1
frameshifting could be performed by S.solfataricus ribo-
somes, mRNAs obtained by in vitro transcription of the
cloned wild-type fucA1 gene and the mutants thereof were
used to program an in vitro translation system prepared as
described by Condo
`et al. (28). To this aim, a promoter of
T7 polymerase was inserted ahead of the gene of interest to
obtain RNA transcripts endowed with the short 50-
untranslated region of 9 nt observed for the natural fucA1
mRNA (24). Autoradiography of an SDS–PAGE of the trans-
lation products (Figure 7) revealed that the wild-type fucA1
transcript produced a tiny but clear band whose molecular
weight corresponded to that of the full-length Ssa-fuc
obtained by site-directed mutagenesis (24); the latter was
translated quite efficiently in the cell-free system in spite of
being encoded by a quasi-leaderless mRNA. Judging from
the relative intensity of the signals given by the translation
products of the wild-type fucA1 and the full-length mutant
fucA1
A
, the efficiency of the 1 frameshifting in the homo-
logous system was 10%. No signals corresponding to the
polypeptides expected from the separated ORFs SSO11867
and SSO3060 (9.6 and 46.5 kDa, respectively) were
observed. However, it should be noted that the product of
SSO11867, even if synthesized, is too small to be detected
in the gel system employed for this experiment. The larger
product of ORF SSO3060, on the other hand, is certainly
absent. These data unequivocally demonstrate that the
ribosomes of S.solfataricus can decode the split fucA1 gene
by programmed 1 frameshifting with considerable effici-
ency producing a full-length polypeptide from the two
ORFs SSO11867 and SSO3060.
Remarkably, under the same conditions at which fucA1
drives the expression of the full-length protein, we could
not observe any product from the fucA1
sm
and fucA1
tm
con-
structs. These data demonstrate that the integrity of the hep-
tanucleotide is essential for the expression of the fucA1 gene
in S.solfataricus, thus further confirming that the gene is
decoded by programmed 1 frameshifting in this organism.
In addition, the lack of expression of fucA1
sm
by translation
in vitro in S.solfataricus contrasts with the efficient
expression of this mutant in E.coli, indicating that the two
Figure 6. Analysis of the expression of fucA1 in different media. (A) Agarose
gel showing the products of RT–PCR encompassing the ORFs SSO11867 and
SSO3060 by using total cellular RNA extracted from cells grown in YSM
(lanes 1 and 2) and YGM (lanes 3 and 4); lanes 1 and 3, control (amplification
of total RNA supplemented with Taq and without reverse transcriptase
enzyme); lane 2 and 4, fucA1.(B) Comparison of the fucA1 mRNA levels in
YSM and YGM by real-time PCR. The inset shows the corresponding
products found in the real-time PCR visualized by ethidium bromide staining.
(C) Western blot of S.solfataricus extracts of cells grown in YSM (lane 2,
80 mg) and YGM (lane 3, 80 mg). Lane 1 recombinant Ssa-fuc (0.2 mg).
Nucleic Acids Research, 2006, Vol. 34, No. 15 4265
organisms recognize different sequences regulating the
translational frameshifting.
DISCUSSION
The identification of genes whose expression is regulated by
recoding events is often serendipitous. In the framework
of our studies on glycosidases from hyperthermophiles, we
identified in the genome of the archaeon S.solfataricus
a split gene encoding a putative a-fucosidase, which could
be expressed through programmed 1 frameshifting (24).
We tackled this issue by studying the expression of fucA1
in S.solfataricus and in E.coli to overcome the problems con-
nected to the scarcity of expression of the a-fucosidase gene
and to the manipulation of hyperthermophiles. As already
reported by others, in fact, it is a common strategy to study
recoding events from different organisms in E.coli (23,33).
The expression in E.coli of the wild-type split gene fucA1
led to the production by frameshifting of two full-length
polypeptides with an efficiency of 5%. This is a value higher
than that observed in other genes expressed by translational
frameshifting in a heterologous system such as the proteins
gpG and gpGT (0.3–3.5%) (33).
The gene fucA1 is expressed in S.solfataricus at very low
level under the conditions tested. In particular, the transcrip-
tional analysis of the gene revealed that it is expressed at very
low level in both YSM and YGM media. Similarly, no differ-
ences in the two media could be found by western blot probed
with anti-Ssa-fuc antibodies, indicating that the low expres-
sion of the enzyme in S.solfataricus is the result of scarce
transcription rather than suppressed translation.
Western blots allowed us to identify a specific band
8.7 kDa heavier than that of the recombinant Ssa-fuc and
experiments of translation in vitro showed that the wild-type
gene expresses a full-length polypeptide exhibiting the same
molecular mass of the recombinant protein. This demon-
strates that the translational machinery of S.solfataricus is
fully competent to perform programmed frameshifting. It
seems likely that the observed discrepancy in molecular
mass might arise from post-translational modifications that
cannot be produced by the translation in vitro. Further experi-
ments are required to characterize the a-L-fucosidase identi-
fied in S.solfataricus.
MALDIMS and LCMSMS analyses of the products in
E.coli of the wild-type split gene fucA1 demonstrated that
two independent frameshifting events occurred in vivo in
the proposed slippery site. In particular, the sequences
obtained by LCMSMS demonstrate that peptide A results
from a simultaneous backward slippage of both the P- and
the A-site tRNAs (Figure 8A). Instead, the sequence of
peptide B is the result of the re-positioning on the 1
frame of only the P-site tRNA; in fact, the next incorporated
amino acid is specified by the codon in the new frame
(Figure 8B). Therefore, the expression by 1 frameshifting
of the wild-type gene fucA1 in E.coli follows the models
proposed for ribosomal frameshifting (34). We confirmed
the significance of the slippery heptanucleotide in promoting
the programmed frameshifting in vivo by mutating the
putative regulatory sequence. The triple mutant fucA1
tm
gave no full-length products; presumably, the mutations in
both the P- and in the A-site of the slippery sequence dramati-
cally reduced the efficiency of the 1 frameshifting as
observed previously in metazoans (35). This result confirms
that the intact slippery sequence in the wild-type gene
fucA1 is absolutely necessary for its expression in E.coli.In
contrast, surprisingly, the single mutant fucA1
sm
showed an
even increased frequency of frameshifting (10%) if compared
to the wild-type and produced only one polypeptide by
shifting specifically in site B. We explained this result obser-
ving that the mutation in the P-site of the slippery sequence
A-AAA-AAT!A-AAG-AAT created a novel slippery
sequence A-AAG identical to that controlling the expression
by programmed 1 frameshifting of a transposase gene in
E.coli (36). Therefore, apparently, the single mutation inacti-
vated the simultaneous P- and A-site tRNA re-positioning
and, in the same time, fostered the shifting efficiency of
the tRNA in the P-site. It is worth noting that, instead, in
S.solfataricus, only the simultaneous slippage is effective
(Figure 8B) and even the single mutation in the slippery
sequence of fucA1
sm
completely annulled the expression of
the gene. This indicates that this sequence is essential in
the archaeon and that programmed frameshifting in
S.solfataricus and E.coli exploits different mechanisms.
Furthermore, since the only difference between the enzymes
produced by the frameshifting sites A and B, Ssa-fuc and
Ssa-fuc
B
, respectively, is the stability at 80C, which is the
S.solfataricus physiological temperature, the functionality of
Ssa-fuc
B
in the archaeon appears questionable.
The reason why fucA1 is regulated by programmed 1
frameshifting is not known. However, the physiological
significance of programmed frameshifting has been assigned
to a minority of the cellular genes while for most of them it
is still uncertain [see (4) and reference therein; (16)]. This
mechanism of recoding is exploited to set the ratio of two
polypeptides such as the tand gsubunits of the DNA
polymerase III holoenzyme in E.coli (12). Alternatively,
programmed frameshifting balances the expression of a
protein, as the bacterial translational release factor 2 and
the eukaryotic ornithine decarboxylase antizyme [see (4)
and (18) and references therein]. In the case of fucA1, the
polypeptide encoded by the smaller ORF SSO11867 could
never be detected by western blots analyses. In addition,
the modelling of Ssa-fuc on the high-resolution crystal
structure of the a-L-fucosidase from Thermotoga maritima
(25,37) showed that the fucA1 N-terminal polypeptide is
not an independent domain. Moreover, we have shown
recently that SSO11867 includes essential catalytic residues
(27), excluding the possibility that a functional a-fucosidase
can be obtained from the ORF SSO3060 alone. There-
fore, several lines of evidence allow us to exclude that
Figure 7. In vitro translation. Of each sample 15 ml was loaded on 12.5%
acrylamide–SDS gel and the newly synthesized proteins were revealed by
autoradiography. Lane 1, no mRNA added; lane 2, fucA1
sm
; lane 3, wild-type
fucA1; lane 4, full-length fucA1
A
; lane 5, fucA1
tm
.
4266 Nucleic Acids Research, 2006, Vol. 34, No. 15
programmed 1 frameshifting is used to set the ratio of two
polypeptides of the a-fucosidase from S.solfataricus. More
probably, this translational mechanism might be required to
control the expression level of fucA1.
Noticeably, this is the only fucosidase gene expressed by
programmed 1 frameshifting. Among carbohydrate active
enzymes, the only example of expression through this recod-
ing mechanism is that reported for a gene encoding for a
a(1,2)-fucosyltransferase from Helicobacter pylori that is
interrupted by a 1 frameshifting (38). In this case, the
expression by programmed frameshifting would lead to a
functional enzyme synthesizing components of the surface
lipopolysaccharides to evade the human immune defensive
system. It is hard to parallel this model to fucA1. Neverthe-
less, the monosaccharide fucose is involved in a variety of
biological functions (39). Therefore, the a-L-fucosidase
might play a role in the metabolism of fucosylated oligosac-
charides; experiments are currently in progress to knockout
the wild-type fucA1 gene and to insert constitutive functional
mutants of this gene in S.solfataricus.
FucA1 is the only archaeal a-L-fucosidase gene identified
so far; hence, it is probably the result of a horizontal gene
transfer event in S.solfataricus. However, since there are no
a-fucosidases genes regulated by programmed frameshifting
in Bacteria and Eukarya, it is tempting to speculate that
this sophisticated mechanism of translational regulation pre-
existed in S.solfataricus and it was applied to the fucosidase
gene for physiological reasons. The identification of other
genes interrupted by 1 frameshifts in S.solfataricus would
open the possibility that they are regulated by programmed
1 frameshifting. Recently, the computational analysis of
prokaryotic genomes revealed that seven Archaea harbour
interrupted coding sequences, but S.solfataricus is not
included in this study (40). A computational analysis on sev-
eral archaeal genomes revealed that 34 interrupted genes are
present in the genome of S.solfataricus, 11 of these genes
are composed by two ORFs separated by 1 frameshifting
and could be expressed by recoding (B. Cobucci-Ponzano,
M. Rossi and M. Moracci, manuscript in preparation).
We have experimentally shown here, for the first time, that
programmed 1 frameshifting is present in the Archaea
domain. This finding is the missing piece in the puzzle of
the phylogenetic distribution of programmed frameshifting
demonstrating that this mechanism is universally conserved.
ACKNOWLEDGEMENTS
We are grateful to Maria Carmina Ferrara for technical
assistance in the real-time PCR experiments and to Maria
Ciaramella and Massimo Di Giulio for useful discussion. We
thank the TIGEM-IGB DNA sequencing core for the sequenc-
ing of the clones. The IBP-CNR belongs to the Centro
Regionale di Competenza in Applicazioni Tecnologico-
Industriali di Biomolecole e Biosistemi. P.P., M. R. and
M. M. were supported by MIUR project ‘Folding di proteine:
l’altra meta
`del codice genetico’ RBAU015B47. This
work was partially supported by the ASI project MoMa n.
1/014/06/0. Funding to pay the Open Access publication
charges for this article was provided by MIUR project
RBAU015B47.
Conflict of interest statement. None declared.
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