From the
Messenger RNA primary structures responsible for translational
efficiency of a photosystem I gene, psaDb, of Nicotiana
sylvestris were studied using a transgenic tobacco system. The
entire 5`-leader (23 base pairs) with the first four amino acid codons
of the protein coding region was fused in frame with the
Translation is a potential step to regulate gene expression, but
its significance is quite poorly understood compared with
transcriptional regulation
(1) . In eukaryotes, the most
extensively studied cases involving translational regulation are the
mammalian genes for ferritin and 5`-aminolevulinate synthase, whose
products are involved in the storage and use of iron, respectively.
Messenger RNAs of these genes have an iron responsible element within
their untranslated leader
(2) . When iron is depleted, this
element is bound by iron responsible element-binding
protein
(3) , which depresses
translation
(2, 4, 5, 6, 7) .
Messenger RNAs of some plant viruses are translated with greater
efficiency than host mRNAs. This phenomenon is ascribed to their leader
sequences. This is true for tobacco mosaic virus, alfalfa mosaic virus
RNA 4, brome mosaic virus RNA 3, potato virus X, and tobacco etch virus
(8). In these cases the viral leaders contain translational enhancers;
however, the underlying mechanisms remain to be elucidated. As for
cellular mRNAs, there have been few reports on translational enhancer
elements to date. In this study, we examined the 5`-portion of
psaDb mRNA (see below) and found that its 5`-leader greatly
enhances the translational efficiency of a chimeric
psaDb is a nuclear gene for a photosystem I (PSI) subunit
in Nicotiana sylvestris(9) , and its product, a PSI-D
subunit, is known to associate with ferredoxin, which is a soluble
electron carrier reduced by PSI. Recently, the untranslated leader of
the Arabidopsis ferredoxin gene, fedA, was shown to
enhance the expression of a reporter gene in a transient expression
assay, but it is not known whether this enhancement occurs at
translation or not
(10) . In this study, we found that the
fedA and psaDb leaders share common motifs and that
mutations introduced into these motifs abolished the translational
enhancer activity of the psaDb leader.
This study clearly showed that the 5`-terminal 35 bases of
the psaDb mRNA contain a translational enhancer element
(Fig. 4), and that base substitution within the 5`-leader
abolished this enhancer activity (Fig. 6). These results lead us
to conclude that the 5`-leader of psaDb contains a
translational enhancer element. This element enhances translation as
much as 20-fold (Fig. 6). Since translational enhancer elements
have rarely been documented in eukaryotic cellular messages,
characterization of this element deserves much attention. The primary
question is how this element activates translation. In recent years,
cap-independent translation initiation mediated by the 5`-leader has
been reported in poliovirus
(16) , encephalomyocarditis
virus
(17) , the human immunoglobulin heavy-chain binding protein
(BiP) gene
(18) , and Antennapedia of
Drosophila(19) . In these cases, an internal ribosomal
entry site within the leader is a determinant for the rate of
translation initiation. However, in contrast to the short psaDb leader of 23 bases, these leaders are generally very long (several
hundred nucleotides or more). Whether the translational enhancer of the
psaDb leader exerts its function in a cap-dependent or
independent manner should be examined. Computer analysis of only the
35-base psaDb sequence using an energy-minimizing algorithm
indicates that it does not form a stable secondary structure (data not
shown). It is possible, though unlikely, that the psaDb leader
sequence in the chimeric CaMV::psaDb-GUS` construct interacts
with the GUS coding sequence to form a secondary structure of
functional significance. To study this, a chimeric construct using a
reporter gene other than GUS is currently being examined.
This study
revealed that the psaDb leader shares common motifs, LM1 and
LM2 (Fig. 5), with the leader of the Arabidopsis ferredoxin gene, fedA. The fedA leader was
reported to enhance the expression of a chimeric reporter gene 25-fold
in a transient expression assay, but it is not known if this
enhancement was exerted transcriptionally or
post-transcriptionally
(10) . Judging from the result that
substitution of LM1 and LM2 in the psaDb leader abolished the
translational enhancer activity (Fig. 6), LM1 and/or LM2 are
integral parts of the translational enhancer. Therefore, it is likely
that LM1 and/or LM2 in the leaders of both psaDb and fedA enhance translation. Since the psaDb product (a PSI-D
subunit) is the binding site of ferredoxin on PSI's
surface
(20) , it is reasonable that psaD and fedA are co-regulated, in this case at translation. Further dissection
of the LM1 and LM2 motifs in respect to their enhancer activity and to
their possible interactions with cellular proteins may provide insight
into translational regulation in plant cells.
The GUS activity/GUS
mRNA value was 5-fold higher in the CaMV::GUS` construct than in the
original CaMV::GUS construct (Fig. 4). This difference is
probably, at least in part, due to the two ATG codons in the 12 amino
acids introduced at the N terminus of the protein, since the number of
N-terminal methionines is known to affect translational
efficiency
(21, 22) . In plant genes, AACAATGGC is highly
conserved at the translation initiation site, and a G residue at the
+4-position (relative to the A of the ATG) is especially
conserved
(23, 24) . In this respect, CaMV::GUS` has a
better context (ATGG) than the original CaMV::GUS (ATGT), and this may
be another contributing factor for the noted difference in
translational efficiency. In addition, the 12 amino acids introduced at
the GUS` N terminus may slightly alter the protein stability and/or
enzyme activity. However, though worthy of study, this difference in
translational efficiency between CaMV::GUS` and CaMV::GUS is not the
main concern of this paper; the enhancer activity of the psaDb leader is.
Last, we mention the applicability of the psaDb enhancer to plant gene technology. In transgenic tobacco, apparent
GUS activity of the CaMV::psaDb-GUS` construct was about 2
orders of magnitude higher than that of the CaMV::GUS construct
(Fig. 2). For what we have examined, the expression of the
CaMV::psaDb-GUS` construct showed no apparent organ
specificity.
We thank Dr. A. Kato of the Hokkaido Agricultural
Experimental Station for advice in tobacco transformation studies, Dr.
J. Maune for critical reading of this manuscript and useful
discussions, and M. Ohtaki for help with some experiments.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-glucuronidase (GUS) gene under the control of the 35 S promoter
of cauliflower mosaic virus (CaMV). This construct
(CaMV::psaDb-GUS`) was introduced into tobacco. GUS activity
and GUS mRNA levels were determined for individual transformants,
revealing that the insertion of the psaDb sequence greatly
enhanced the GUS activity relative to GUS mRNA abundance. The GUS
activity/GUS mRNA was 14 times higher in the CaMV::psaDb-GUS`
transformants than in the control CaMV::GUS` transformants. The high
GUS activity/GUS mRNA of the CaMV::psaDb-GUS` transformants
was reduced 20-fold when 13 bases within the psaDb leader were
altered. These 13 bases are common to the leaders of an Arabidopsis ferredoxin gene and the psaDb gene of N.
sylvestris. Since GUS proteins encoded by these chimeric GUS genes
have identical amino acid sequences, these results indicate that the
5`-leader of the psaDb mRNA contains a translational enhancer
element.
-glucuronidase
(GUS)
(
)
reporter gene in vivo.
Materials
N. sylvestris is a diploid
ancestor of Nicotiana tabacum, which is an
amphidiploid
(11) . The two species have highly homologous
genomes. Constructs containing fragments from an N. sylvestris gene, psaDb(9) , were transformed into N.
tabacum.
Construction of Chimeric GUS Genes
Four fusion
genes (see Fig. 1and Fig. 6) were used in this study. 1)
pBI121 contains the CaMV::GUS construct
(12) . 2) For
CaMV::psaDb-GUS` two oligonucleotides
(5`-CTAGACTTCTCTCAATCCAACTTTTCTATGGCCATGGCAC-3` and
5`-GATCGTGCCATGGCCATAGAAAAGTTGGATTGAGAGAAGT-3`) were annealed and
inserted into the XbaI-BamHI site of pBI221. After
sequencing the introduced region, an EcoRI-HindIII
fragment containing the resultant chimeric gene was introduced into a
binary vector, pBI101, in place of the original promoterless GUS gene
of pBI101
(12) . 3) For CaMV::GUS` two oligonucleotides
(5`-CTAGATGGCTATGGCTC-3` and 5`-GATCGAGCCATAGCCAT-3`) were annealed and
introduced into pBI101 as for CaMV::psaDb-GUS` described
above. 4) For CaMV::psaDbLM-GUS` two oligonucleotides,
(5`-CTAGACTTCGAGACCTCACCAGGGTCTATGGCCATGGCAC-3` and
5`-GATCGTGCCATGGCCATAGACCCTGGTGAGGTCTCGAAGT-3`) were annealed and
introduced into pBI101 as described above.
Figure 1:
Constructs of
the chimeric GUS genes. A, the 5`-terminal sequence of the
psaDb mRNA. The transcription start site is shown as
+1. Arrows indicate palindromic sequences.
B, schematic representation of the chimeric GUS genes.
Arrows indicate the transcription start sites. CaMV::GUS
represents the original GUS reporter gene of pBI121 (12). The psaDb fragment shown in A was inserted into the untranslated
leader of CaMV::GUS to form CaMV::psaDb-GUS`. CaMV::GUS` is a
control gene identical to CaMV::psaDb-GUS` except for the lack
of the 23-base pair psaDb leader sequence and the two base
substitutions as shown in C. C, nucleotide sequences
of the chimeric GUS genes downstream of the XbaI sites
(TCTAGA), aligned with the N-terminal sequences of the encoded
proteins. The underlined portion shows the inserted psaDb sequence. Arrowheads indicate the base substitutions to
alter the palindromic sequences.
Figure 6:
Substitution of LM1 and LM2 decreases
translational efficiency. A, the indicated 5`-leader motifs
LM1 and LM2 in the CaMV::psaDb-GUS` construct were altered
(shadedboxes) to generate a new chimeric construct,
CaMV::psaDbLM-GUS`. B, GUS activity/GUS mRNA values
were determined for individual transformants of
CaMV::psaDb-GUS` and CaMV::psaDbLM-GUS`, and the
averages are shown.
Ti-Mediated Gene Transfer and GUS Assays
Binary
vectors containing the chimeric GUS genes mentioned above were
mobilized into Agrobacterium tumerfaciens LBA4404, and leaf
discs of N. tabacum cv. Petit Havana SR1 were
transformed
(13) . Plants were grown at 25 °C under
continuous light. From the regenerated transformants (M generation), fully developed leaves of 3-6 cm in Magenta
culture boxes (Magenta GA-7, Sigma) were harvested and frozen in liquid
N
, ground to fine powder, and stored at -80 °C. A
part of the powdered tissue was used for GUS assays
(12) , and
the rest was used for RNA extraction. Protein concentration was
determined according to Bradford
(14) and used to normalize
measured GUS activities.
RNA Analysis
Total RNA was extracted from the
powdered tissue by the acid guanidium thiocyanate-phenol-chloroform
method
(15) , followed by three phenol/chloroform extractions and
LiCl precipitation. A GUS gene-specific primer
(5`-GTCGAGTTTTTTGATTTCACGGGTTGGGGTTTCTA-3`) was labeled at the 5`-end
with P and used for primer extension analysis as described
previously
(9) . Extension products were electrophoresed in 8
M urea, 6% polyacrylamide gel, and the radioactivity of each
band was analyzed using a Fujix BAS2000 Imaging Analyzer (Fuji Photo
Inc., Japan).
Experimental Strategy
psaDb mRNA has a
23-base untranslated leader (Fig. 1A). In this study, we
examined the possible involvement of this leader in translational
regulation of psaDb mRNA by use of a transgenic tobacco
system. The entire 5`-leader with the initial four codons of psaDb (Fig. 1A) were introduced into the untranslated
leader of the original CaMV::GUS construct of pBI121 to obtain a
chimeric GUS gene named CaMV::psaDb-GUS` (Fig. 1, B and C). As a result of this insertion, the GUS protein
gained 12 amino acids at its N terminus (Fig. 1C,
CaMV::psaDb-GUS`), which might alter the specific activity or
turnover rate of the protein. We refer to this modified GUS protein as
GUS`. In order to assess the effect of the inserted psaDb sequence on GUS` gene expression, we prepared a control GUS`
construct, CaMV::GUS`, whose protein coding region has the same
nucleotide sequence as that of CaMV::psaDb-GUS` except for the
sixth and the twelfth nucleotides from the initiator ATG
(Fig. 1C, arrowheads). These nucleotides were
substituted in CaMV::GUS` to alter the palindromic sequences at the
translation initiation site but did not affect the amino acid sequence.
These three types of chimeric genes, CaMV::GUS,
CaMV::psaDb-GUS`, and CaMV::GUS`, were introduced into
tobacco, and the resultant transformants were regenerated.
5`-Terminal 35 Bases of psaDb mRNA Enhance Translational
Efficiency in Vivo
The steady-state GUS activity in leaves was
determined for individual transgenic plants grown under continuous
light (Fig. 2). In comparison with the control transformants
(CaMV::GUS`), GUS activity was much higher in the transformants having
the psaDb sequence (CaMV::psaDb-GUS`). This
enhancement of GUS activity may have been caused by internal
cis-elements for transcriptional activation, stabilization of
the transcripts, and/or elevation of translational efficiency. For the
purpose of comparing the translational efficiencies among these
transgenic plants, we determined the steady-state mRNA levels of the
chimeric GUS genes by primer extension analysis (Fig. 3). The
lengths of the extension products were different according to the
lengths of the inserted fragments (Fig. 3). GUS activities of the
individual transformants were divided by their respective mRNA levels
(Fig. 4, GUS activity/mRNA), to examine the
translational efficiency of the given GUS mRNAs. This value was 14
times higher in CaMV::psaDb-GUS` than in CaMV::GUS`. Thus, the
inserted psaDb leader of 23 bases and/or the palindromic
sequences at the translational initiation site (Fig. 1, A and C) greatly enhances the translation of GUS` messages.
Figure 2:
Insertion of the psaDb sequence
elevates GUS activity in transgenic tobacco. Mature leaves of
individual transgenic lines were assayed for GUS activity. Note the
logarithmic scale.
Figure 3:
mRNA level of the chimeric GUS genes in
transgenic tobacco. Total RNAs prepared from an untransformed plant
(SR1) and from the transformants indicated were subjected to primer
extension analysis. Numbers indicate transgenic lines as in
Fig. 2. Arrowheads indicate the bands of chimeric GUS mRNAs,
which are absent from the untransformed plant
(SR1).
Figure 4:
The ratio of GUS activity to GUS mRNA. The
average value of the CaMV::GUS transformants is presented as 1.0.
A, the GUS activity/GUS mRNA was determined for each
transformant. Numbers indicate transgenic lines as in Figs. 2
and 3. B, average values and standard deviations for each of
the chimeric constructs are presented.
5`-Leader Motifs Are Shared between psaDb of N.
sylvestris and fedA of Arabidopsis
Recently, Casper and Quail
(10) reported that the 5`-untranslated region (UTR) of the
Arabidopsis ferredoxin gene, fedA, enhances gene
expression. They fused the 5`-UTR of fedA to a luciferase
reporter gene under the control of the fedA promoter, and the
expression of this fusion gene was examined by a transient expression
assay using Arabidopsis seedlings. When the 5`-UTR was
deleted, luciferase activity was reduced 25-fold. The apparent
similarity of the 5`-UTRs of psaDb and fedA in
enhancing the reporter activity of chimeric genes led us to speculate
that the translation of both genes may be enhanced by similar
mechanisms. Therefore, the 5`-leader sequences of psaDb and
fedA were compared (Fig. 5). These leaders share two
common motifs, and these motifs cover more than half of the psaDb leader. We designate these 5`-leader motifs LM1 (TCTCAA) and LM2
(CAACTTT).
Figure 5:
Comparison of the entire 5`-leader
sequences of the psaDb gene of N. sylvestris and the
fedA gene of Arabidopsis. Two conserved motifs
designated as LM-1 and LM-2 are
underlined.
Substitution of LM1 and LM2 within CaMV::psaDb-GUS`
Greatly Decreases Translational Efficiency
In order to clarify
whether LM1 and LM2 really operate as translational enhancers, these
two motifs within the CaMV::psaDb-GUS` construct were altered
to form CaMV::psaDbLM-GUS` (Fig. 6A). This
construct with leader mutations was introduced into tobacco, and its
GUS activity/GUS mRNA was determined for each transformant. As shown in
Fig. 6B, mutations in LM1 and LM2 reduced the
translational efficiency of the psaDb-GUS` message 20-fold.
This indicates that LM1 and/or LM2 are integral parts of the
translational enhancer.
(
)
Thus, the
CaMV::psaDb-GUS` construct could be used instead of CaMV::GUS
for a 100-fold increase of expression. Furthermore, the psaDb leader itself could be used to increase expression levels for a
wide variety of plant vectors.
-glucuronidase; PSI, photosystem I; CaMV,
cauliflower mosaic virus; UTR, untranslated region.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.