In vivo evaluation of the context sequence of the translation
initiation codon in plants
Marcin Lukaszewicz
a,b,1, Marc Feuermann
b,1, Be´ne´dicte Je´rouville
b, Arnaud Stas
b,
Marc Boutry
b,*
aInstitute of Microbiology,Wroclaw Uni6ersity,Przybyszewskiego63,51-148Wroclaw,Poland
bUnite´ de Biochimie Physiologique,Uni6ersite´ Catholique de Lou6ain,Place Croix du Sud2-20,B-1348Lou6ain-La-Neu6e,Belgium
Received 8 October 1999; received in revised form 3 December 1999; accepted 3 January 2000
Abstract
Statistical analysis of the AUG initiation codon context in several plant organisms identified a nucleotide preference in some positions around the AUG. Sixteen AUG contexts were studied using transient expression in tobacco, maize and Norway spruce. Besides the importance of A or G at position −3, we revealed the role of positions −2, −1 for which AA or CC were found to be the best for tobacco and maize, respectively. GC (positions+4, +5) were also found to be important in both tobacco and maize. Finally, we identified a variation in context efficiency according to cell type, since A was better than G at position −3 in tobacco leaf protoplasts, while both nucleotides were equally efficient in tobacco suspension cells. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Consensus AUG context; Dicot; Gymnosperm; Monocot; Plant; Translation context; Translation initiation
www.elsevier.com/locate/plantsci
1. Introduction
In eucaryotes, translation by cytosolic ribo-somes generally occurs at the first transcript AUG. However, efficient recognition of an AUG codon as a translation initiation site depends on several factors, such as the distance from the transcript 5%
end, the secondary structure around the AUG codon, and the nucleotide sequence flanking the translation initiation site [1 – 3]. The frequency of nucleotides surrounding the initiation AUG (con-text) has been extensively analysed in genes avail-able in databases [4], revealing a consensus sequence (i.e. the most frequent nucleotides) dis-tinct for various groups of organisms. Thus Ya-mauchi [5] deduced the consensus sequence for protozoa as U A A A AUG A N A U, with A at
position −3 being the best conserved nucleotide. A at position −3 was also shown to be a con-served nucleotide in Saccharomyces cere6isiae [6]. From the studies of 699 vertebrate AUG contexts, Kozak [7] deduced C A/G C C AUG G to be a consensus sequence. Finally, Lu¨tcke et al. [8], Joshi [9] and Cavener and Ray [10] made a similar analysis for plant genes. Joshi [11] recently ex-tended this analysis to 5074 sequences allowing identification of a consensus context for monocot (a/c A/G A/C c AUG G C) and dicot plants (a A A/C a AUG G C). In most cases, adenine at −3 and guanine at +4 position were found to be the most frequent nucleotides.
The biological significance of the consensus se-quence derived from statistical analysis of data-bases is less documented, especially in plants. Lu¨tcke et al. [8] tested the AUG context in both the rabbit reticulocyte and the wheat germ transla-tion systems. Substituting A at positransla-tion −3 by G, U or C significantly reduced expression in the
* Corresponding author[f1]. Tel.: +32-10-473621; fax: + 32-10-473872.
E-mail address:[email protected] (M. Boutry)
1These two authors contributed equally to this work.
rabbit reticulocyte system, which was not the case in the wheat germ system, suggesting a possible difference in the mechanism of translation initia-tion between plants and animals. Systematic muta-genesis experiments with the vertebrate preproinsulin gene expressed in transfected animal cells confirmed the importance of purine at posi-tion −3, as well as the importance of C at posi-tions −2 and −1 and G at position +4 [12,13]. More recent in vitro and in vivo studies with influenza and parainfluenza viral RNA also sug-gested the importance of positions +4, +5 and
+6 [14,15]. However, the role of the +5 and +6 positions was questioned recently by Kozak [16]. Experimental data also supported the importance of A at position −3 in Saccharomyces cere6isiae
[17]. Transient expression in tobacco mesophyll protoplasts of the b-glucuronidase reporter with two different AUG contexts: CCACC AUG G (as an ‘optimal’ sequence for the rat preproinsuline gene in COS cells) and AACA AUG G (as a plant consensus context) did not show any significant difference in translation efficiency between the two constructs [18]. Luehrsen and Walbot [19] studied the effect of an upstream out-of-frame AUG codon that severely affected expression of the re-porter gene, even though it was surrounded by a poor context, suggesting a lesser role for AUG context in plants than in animals. On the other hand, the importance of AUG context in plants was supported by a study showing a 4-fold im-provement of translation of a chitinase protein when positions −3 and +4 were modified into A and G, respectively [20]. Similar results were ob-served for two viral RNA [21,22] and the GUS reporter gene [23].
Much less is known about the impact of other positions and the interaction between them. Thus, it is impossible to predict how efficiently a tran-script with an AUG initiation context different from the consensus will be translated. Another interesting issue is how the consensus between organisms diverged during evolution. For in-stance, does the distinct consensus determined for the dicot and monocot plants reflect a modifica-tion in the translamodifica-tional machinery? In this study, we have addressed these issues by comparing luci-ferase (LUC) expression produced from 16 gene constructs with distinct AUG contexts introduced
in Nicotiana tabacum, Zea mays and Picea abies
cells.
2. Methods
2.1. AUG context database research
In April 1996, we retrieved sequences from the GenBank-databases. The sequence description was limited to definition, accession number, references, expression information (developmental stage and tissue type), leader length and sequence plus six nucleotides after the AUG, considered by submit-ting authors as the initiator. Redundancies (for example cDNA and genomic sequence) were elimi-nated by looking for sequences with the last 16 nucleotides identical. The final database of 2190 genes was subdivided into three databases contain-ing dicot (1533), monocot (624) and others, mainly conifer, (33) plant genes.
We retrieved records from sequence databases by key word searches. We used the following query:
((plantae [src] NOT thallobionta) AND (mRNA [fea] OR CDS [fea] OR UTR [fea])) NOT (chloro-plasts OR mitochondri*)
A total of 7861 sequences were retrieved. The sequences were edited using Word for Windows 6.0. Several macro definitions were written in WordBasic in order to compile and analyse the sequences. Entries without the AUG initiation codon, and not preceded by at least one nucleotide were deleted. We used the Cavener 50/75 rule [4] to describe the consensus.
2.2. DNA contructs
Using as template the luc gene [24] cloned into the HindIII-SacI restriction sites of plasmid Blue-script SK+, we amplified the 5%region oflucwith the primers 5%
CCGAAGCTTGGATCCCTC-GAGGAAGACGCCAAAAAC 3% and 5% TG-GATAGAATGGCGCCG 3%. The first primer
introduced the HindIII, BamHI and XhoI sites in front of the coding sequence of luc and removed the ATG initiation codon. The second primer replaced T, at position +48, by A in order to remove the XbaI site at this position without changing the corresponding amino-acid sequence. The amplified fragment was cut with HindIII (5%
wholeluccoding sequence without the ATG initia-tion codon was then substituted for the gusAgene in the 35SGUS construct described in Lukaszewicz et al. [25] and cut with BamHI and SacI. This construct, thus containing the luciferase gene with-out ATG initiation codon and under the control of the CaMV 35S promoter and nos terminator, was used as a negative control (construct 17).
To prepare constructs 1 – 16, two primers were first synthetised for each context: 5%GAATTCX1X2X3X4ATGX5X6G 3% and 5% GATCCY6Y5CATY4Y3Y2Y1G 3% where X1 – 6 and Y6 – 1correspond to coding and non-coding strands of the chosen context. After hybridization, both primers were introduced between the BamHI and XhoI restriction sites of construct 17. At least two independent plasmid preparations for each con-struct were used.
2.3. Transient gene expression in tobacco using
protoplast electroporation
Protoplasts were prepared from young tobacco cv SR1 leaves and electroporated as described in Lukaszewicz et al. [25].
2.4. Transient gene expression using biolistic
transformation
Five hundred microlitre aliquots of tobacco BY2 [26], maize BMS [27] or Norway spruce embryogenic cells (provided by Dr J.-L. Fourre´, Unite´ des Eaux et Foreˆts, Universite´ catholique de Louvain, Louvain-la-Neuve) were spread on solid MS medium [0.44% Murashige and Skoog [28] salts (ICN), 1% agar and 3% sucrose]. The pH was modified to 5.75 before autoclaving. Growth oc-curred at 26°C in darkness for 24 h.
Biolistic transformation was performed using the PDS-1000/He System (BIO-RAD) as described by Lukazsewicz et al. [25].
2.5. Luciferase assays
After 16 h incubation, electroporated proto-plasts were collected by centifugation at 700 rpm (Sorvall HL-4) for 6 min and resuspended, as described by Luehrsen and Walbot [29], in 500 ml of Cell Culture Lysis Reagent (CCLR: 100 mM potassium phosphate, pH 7.8, 1 mM EDTA, 7 mM b-mercaptoethanol, 10% glycerol) and frozen
in liquid nitrogen. To perform firefly luciferase (LUC) and control Renilla luciferase (RUC) as-says, samples were thawed and sonicated for 15 s (Virsonic Cell Disrupter 16-850; Virtis, Gardiner, NY, set at 35% power). After sonication, extracts were centrifuged for 15 min at 15 000 rpm, and the supernatant tested for LUC and RUC activity.
Bombarded tobacco BY2 cells, maize BMS cells, and Norway spruce embryogenic cells, as well as young tobacco SR1 leaves, were collected in microtubes and resuspended in 500ml of CCLR and ground. The extracts were centrifuged for 15 min at 15000 rpm, and the supernatant tested for LUC and RUC activity.
LUC and RUC activity was measured by lumi-nometry on a Lumat LB 9501 (Berthold GmbH & Co., Wildbad, Germany). To test LUC activity, extracts (10 ml) were placed in luminometer tubes to which 50 ml of Luciferase Assay Reagent (LAR, Promega: 20 mM Tricine, pH 7.8, 5 mM MgCl2, 0.1 mM EDTA, 3.3 mM dithiothreitol, 270 mM Coenzyme A, 500 mM luciferin, 500 mM ATP) were automatically injected. Photons were counted for 6, 2 s after injection. For RUC activity, ex-tracts (10 ml) were placed in luminometer tubes to which 50 ml of Matthews buffer (0.1 M Na2HPO4/ KH2PO4, pH 7.6, 0.5 M NaCl, 1 mM EDTA, 0.02% BSA) containing coelenterazin (10 mM) were automatically injected. Photons were counted for 6 s, 2 s after injection.
We checked whether luciferase activity was lin-ear with the protein amount. The relative luci-ferase activity was calculated as the ratio between the test LUC and the control RUC activity.
2.6. Statistics
The number of independent transformations was as follows: seven for tobacco leaf protoplast electroporation, six for tobacco leaf ment, eight for tobacco suspension cell bombard-ment, 12 for maize suspension cell bombardment (with additionnal ten bombardments for con-structs 12, 13 and 14) and finally ten for Norway spruce suspension cell bombardment. Two enzyme assays were performed on each tobacco leaf proto-plast electroporation and one for each bombardment.
the most expressed construct for each plant type studied. We also calculated the probability of the values obtained being significantly different using the Student law. We considered as significantly different those pairs of values 5% or less likely to share a same group.
3. Results
3.1. Nucleotide frequencies of the translation
initiation context in different tissue types
The starting point for this work was the identifi-cation of small open reading frames within the leader of several transcripts encoding a plasma membrane H+-ATPase [25,30]. We obtained evi-dence that these open reading frames were trans-lated by a fraction of the scanning ribosomes, although they did not have an AUG context close to the consensus. This prompted us to examine the AUG context of plant genes. We therefore re-trieved the AUG context from the genes available in the databases. Using the 50/75 rule introduced by Caverner [4], we deduced the consensus con-texts as a A A/C a AUG G C and c A/G C/A C AUG G C for dicot and monocot plants, respec-tively. As these data were largely confirmed by a recent study [11], we will not comment further on them.
3.2. Selection of AUG contexts for experimental
e6aluation
To experimentally evaluate the importance of the AUG context, we chose the firefly luciferase (luc) gene [24] as a reporter. The sequence sur-rounding the initiation AUG was replaced with a cassette allowing an exchange of 16 different AUG contexts (Table 1). As a negative control, we used a construct with the deleted AUG initiator codon (construct 17). Besides the comparison of monocot and dicot consensus contexts, we considered the importance of the residues at positions −3, −2,
−1 and +4, +5 using the dicot consensus as a starting point (Table 1). We also calculated the frequency of each of the 16 contexts in both dicot and monocot databases (Table 1). It appears that the consensus contexts derived for dicots are in-deed the most frequent of the 16 contexts used in this study. Concerning the monocot database, two
contexts (constructs 6 and 7) that are frequent in dicot genes, are as frequent or more frequent than the consensus sequences derived for monocots. This indicates that the consensus context, which is a juxtaposition of the most frequent nucleotide(s) at each position, is not necessarily the most fre-quent context. The 16 sequences tested here corre-spond to 13.9% of the genes in the dicot database and 9.3% of the genes in the monocot database. Although in vitro translation systems have been used to study the cell translation machinery, they might not reflect the real conditions and organisa-tion of the translaorganisa-tion system [21,31] and might therefore introduce some bias in the expression of the test constructs.
Transgenic plants, in which the test gene is integrated in the genome, would not be convenient for a quantitative comparison between several constructs because the so-called ‘position effect’ would introduce expression variations that might be much larger than the variations associated with the AUG context. In this case, RNA quantifica-tion would also be required, thus introducing a further parameter. We therefore relied on a tran-sient expression system, using either electropora-tion or bombardment. As an internal control, we used a plasmid carrying the Renilla luciferase [32]. The reporter and control luc genes were both placed under the control of the cauliflower mosaic virus 35S promoter.
3.3. Assay of the AUG context in tobacco leaf
protoplasts
In tobacco protoplasts, the highest expression level was obtained with construct 1, which had a consensus sequence for dicots (Fig. 1A, construct 1). A control without AUG had no activity (con-struct 17). The second dicot consensus context (C instead of A at position −2) had a LUC activity significantly (see Section 2 for the definition of this word) reduced by approximately 30% (construct 6). Expression of constructs 2, 3 and 4 revealed that substitution at position −3 of A by any other nucleotide also significantly reduced LUC activity by about one third. The importance of A, and not just as a purine (A or G) as it sometimes appears in consensus sequences [7] at position
A similar significant reduction in expression re-sulted from the substitution of A by C at −2 or
−1 positions (constructs 6 and 5 versus 1). This negative effect was not cumulative when both −1 and −2 positions were substituted by C (con-struct 12).
Changing four nucleotides (−4 to −1) up-stream from the AUG (anti-upup-stream consensus, construct 10) reduced LUC activity more than the substitution of two nucleotides (+4, +5) down-stream from the AUG (anti-downdown-stream consen-sus, construct 11). Combining anti-upstream and anti-downstream consensus sequences resulted in a further significant LUC reduction down to 10% (construct 9). The two monocot consensus con-texts I and II (Table 1) were not favourable in
tobacco, leading to a significant decrease in LUC activity of approximately 25 (construct 12) or 50% (construct 13).
We have also tested two gene contexts without A at −3 and G at +4 (constructs 15 and 16). Construct 15 corresponds to the AUG context found in a nodulin gene specifically expressed in root nodules [33]. Construct 16 corresponds to another tissue-specific gene coding for a proline-rich protein similar to the class of HyPRPs [34]. LUC activity obtained with these two constructs was similar to that obtained with the anti-consen-sus (construct 9), suggesting that these alternative contexts are not appropriate, at least for the leaf mesophyll cells. These three contexts are also very rare in vertebrate mRNA [10].
Table 1
Sequence, description and frequencies of the 17 AUG contexts tested
Frequency in dicot Frequency in monocot Description
Contruct AUG context
databasea (%) databasea (%)
0
1 AAAA AUG Consensus dicot I 3.914
GC
0 2 AGAA AUG Derives from consensus dicot I (G at−3) 0.783
GC
AUAA AUG 0.065 0
3 Derives from consensus dicot I (U at−3) GC
0.160 4 ACAA AUG Derives from consensus dicot I (C at −3) 0.718
GC
0.160 5 AAAC AUG Derives from consensus dicot I (C at −1) corre- 0.652
GC sponds to consensus monocot III
6 AACA AUG Consensus dicot II (C at −2) 2.609 2.083 GC
AGCA AUG 2.244
7 Derives from consensus dicot II (G at −3) 1.826 GC
0.481 1.370
Derives from consensus dicot II (U at −3)
8 AUCA AUG
GC
UCGU AUG 0
9 Anti-consensus dicot 0
CU
10 UCGU AUG Anti-upstream consensus dicot 0 0 GC
AAAA AUG 0
11 Anti-downstream consensus dicot 0.065 CU
AACC AUG 1.603
12 Consensus monocot I 0.913
GC
AGCC AUG 2.083
13 Consensus monocot II 0.652
GC
0.481 0.130
Derives from consensus monocot (C at −3) 14 ACCC AUG
GC
UCCU AUG Nodulin (genbank L22765) 0.130 0 15
CC
0 16 GUUG Proline-rich protein (genbank L20755) 0.065
AUG AA
Negative control
D(AUG) 17
Fig. 1. Relative LUC activity in extracts from tobacco leaf protoplasts after electroporation (A), tobacco suspension cells after biolistic transformation (B), tobacco leaf cells after biolistic transformation (C), maize suspension cells after bi-olistic transformation (D) and from Norway spruce suspen-sion cells after biolistic transformation. LUC activity of each construct was related to that of construct 1, considered as 100% for tobacco, construct 12 for maize and construct 5 for Norway spruce, respectively. In C, only constructs 1 and 2 were tested.
cells are undifferentiated and actively divide. In this case, the constructs were introduced by biolis-tic methods.
The overall expression pattern (Fig. 1B) was roughly similar to that for leaf except that the dicot consensus I (construct 1) was not so conspic-uous. Constructs 2 and 3 were as efficient. To check whether this difference was due to the trans-formation system (protoplast electroporation ver-sus intact cell bombardment) or rather the plant material (leaf versus culture cells), we also trans-formed tobacco leaves with constructs 1 and 2, using particle bombardment (Fig. 1C). LUC activ-ity was significantly lower for construct 2 in both leaf protoplast electroporation and intact leaf bombardment, while this was not the case for cell culture bombardment. We could therefore at-tribute the variation in the relative expression between constructs 1 and 2 to the plant material. Another significant difference between the sus-pension and leaf cells was the relative greater importance of the GC downstream consensus con-text in the former (compare constructs 10 and 11 of Fig. 1A and B).
3.5. Assay of the AUG context in maize
suspension cells
Although initially designed to be analysed in a dicot species, the various constructs were also introduced by particle bombardment into suspen-sion cells from maize, a monocot species (Fig. 1D). The highest activity was obtained with con-structs 12 and 13, with the monocot consensus sequence I and II, respectively. In contrast to tobacco leaf protoplasts, but as with tobacco sus-pension cells, either A or G at position −3 pro-duced high LUC activity. Another difference was that the presence of C at positions −1 and −2 was necessary to support a high expression level (compare construct 12 and constructs 5 or 6). The lowest expression (B20%) was obtained, as in
tobacco, with constructs 9, 15 and 16. This is not surprising, as the anti-consensus sequence is ap-propriate to both plant classes. Both the anti-up-stream or downanti-up-stream consensus affected LUC activity, significantly reduced to approximately 30%. No significant difference could be found between the other constructs, with an average LUC activity close to 50% of that obtained with constructs 12 or 13.
3.4. Assay of the AUG context in tobacco
suspension cells
3.6. Assay of AUG context in Norway spruce suspension cells
Finally, we bombarded the 17 constructs in embryogenic Norway spruce cell cultures to study the importance of AUG context in a gymnosperm plant. When this study was initiated, only a small number of gymnosperm gene sequences was avail-able in the databases, thus preventing a consensus from being determined. We had therefore no ex-pectations for the relative expression level of the different constructs as was the case for tobacco and maize. However, Joshi et al. [11] deduced a consensus context from 93 non-angiosperm higher plants (bryophytes, pteridophytes and gym-nosperms): A/G a a/c ATG G C. Construct 5, which has a sequence corresponding to this con-sensus, gave the highest level of LUC expression in Norway spruce cells (Fig. 1E). While the anti-dicot consensus (construct 9) produced less than 25% relative LUC activity, the comparison of anti-up-stream (construct 10) and anti-downanti-up-stream (con-struct 11) consensus sequences showed that only the upstream sequence was important.
4. Discussion
Using two different transformation techniques (leaf protoplast electroporation and suspension cell bombardment) we tested the translation effi-ciency of 16 initiator AUG contexts in tobacco. The biolistic technique was also used to test the same constructs in a monocot (maize) and a gym-nosperm (Norway spruce) plant. The transient expression system used in this study was very convenient for the in vivo characterization of the constructs. Since transient expression might enable the expression of several gene copies within a cell, we tested LUC expression using five times less DNA during protoplast electroporation. LUC ac-tivity was reduced accordingly, but the same rela-tive activities were found for the different constructs (not shown), indicating that there was no bias in the data due to a possible saturation effect. The data obtained with the consensus and anti-consensus contexts tested here indicated that the reporter system we used was reliable, at least to some extent.
However, we should be aware that the context surrounding the AUG belongs to an RNA
molecule with a specific leader and overall struc-ture. This might affect the way in which RNA is recognized by the scanning ribosome subunit. Sometimes [35], but not always [25], changing the reporter gene might lead to different results. RNA secondary structures might severely affect transla-tion efficiency. This is especially the case when stem-loop structures are present near the initiation codon [36 – 39]. However, no strong putative sec-ondary structure was predicted around the initia-tion codon in the various constructs used in this work. The length and the nature of the leader might also change the impact of an AUG context. Further work will therefore require other reporter genes as well as different 5% or 3% untranslated
sequences to be tested.
Finally, the transient expression system we used does not represent the optimal in vivo system. Confirming the data with stable transgenic plants would be useful even though in this case the bias associated with the position effect should be taken into account, for instance by RNA quantification. The consensus contexts for dicots as defined recently by Joshi et al. [11] and confirmed here are only represented in B7% of the genes in the
database (1996), indicating that the consensus is nothing more than the juxtaposition of the most frequent nucleotides at different positions. This does not mean that the resulting consensus se-quences are the most frequent contexts. This also indicates that an exhaustive experimental analysis of initiation AUG contexts is not realistic since it should combine all the permutations at −3, −2,
−1 and +4, +5, which results in 1024 possible sequences. However, the 16 sequences tested here correspond to 13.9% of the genes in the dicot database and 9.3% of the genes in the monocot database. This study should therefore be extended by including other moderately frequent contexts. In vitro studies in plants had suggested a less important role for the AUG context in plants [8] than in other organisms. This did not agree with data obtained in vivo showing the importance of
effi-ciency, especially concerning the monocot versus dicot comparisons. Since a single species plant was used for each plant division, testing the same constructs on additional plant species for each group would be required before generalizing the conclusions.
One of the aims of this study was to experimen-tally evaluate the translational efficiency of the consensus obtained from a statistical analysis. The 50/75 rule of Cavener, as used in many publica-tions, creates a complication because it introduces a co-consensus (two nucleotides at one position, for example A/G at −3 position in monocots and A/C at −2 position in both monocots and dicots). Thus, taking into account the −3 to +5 region, there are two consensus sequences for dicots and four for monocots. One consensus sequence derived from dicot plants (AAA AUG GC), in fact, enabled the highest expression in tobacco leaves. This was not the case with the second consensus (ACA AUG GC). The same was true with the monocot consensus I and II (ACC AUG GC and GCC AUG CC) which allowed the highest translation in maize, contrary to the con-sensus III (AAC AUG GC).
In the literature, purines are usually claimed to be important at position −3. This was the case for maize and tobacco suspension cells. However, in tobacco leaf cells, A and not just a purine seems to be important since LUC expression decreased to the same level whether A was changed into G or a pyrimidine.
Although the −3 position is the most con-served upstream of the start codon, our experi-mental data showed that, in dicots, changes at −2 and −1 positions affected translation efficiency at least as much as changes at the −3 position. For monocots, this effect seems to be even more pro-nounced as changing C at −1 and/or −2 posi-tion resulted in an approximately 50% reducposi-tion in translation efficiency. Under our experimental conditions the highest expression was produced by AA in dicots and CC in monocots at −2 and −1 positions.
There is still only a limited number of sequences available in databases from non-angiosperm higher plants. A statistical analysis is therefore not yet possible. We performed expression experiments on Norway spruce belonging to the Pinophyta (gymnosperm) division. The expression pattern was different from that of monocot or dicot cells.
For instance, although GC at positions +4 and
+5 were the most conserved nucleotides after the AUG codon itself [11], the anti-downstream con-text produced one of the highest expressions in Norway spruce.
It is clear from this study that there is no universal context able to produce the highest translation in all plant species. Moreover, we also have to consider that an AUG context might not be the most suitable for all cell types within the plant. The comparison between tobacco leaf and suspension cells gave two examples of significant differences. First, in the suspension cells, a non-consensus sequence (GAA AUG GC) allowed an expression level as high as that obtained for the consensus. Then, in leaf cells the GC downstream context seemed less important than in suspension cells. The statistical study also suggested that the AUG context might be involved in tissue-specific modulation of translation. In fact, the genes ex-pressed in root nodules had a consensus context slightly divergent from that of the general dicot consensus context since U was the most frequent nucleotide at positions −4 and −1 (data not shown). As a whole, these data suggest that the translation efficiency of a given context might vary according to cell type. This was not surprising since, using mutations of the AUG initiation con-text of the alcohol deshydrogenase, Feng et al. [40] revealed a different effect of the AUG initiation context at two different stages (larval and adult) of
Drosophila development.
The observation that sub-optimal AUG con-texts are present in many genes suggests the hy-pothesis that this context might be involved in modulating gene expression. This might be the case for transcripts encoding two proteins that differ at their N-terminal end. For instance, some transcripts encode enzymes that operate both in the cytosol and in an organelle, or in different organelles. The longest protein, initiated at the first AUG in a sub-optimal context, includes a targeting sequence, while the smaller protein, ini-tiated by ribosomes that skipped the first AUG, remains in the cytosol [41].
pro-ton-ATPase [25]. This is a major enzyme in the plasma membrane and it has been suggested that modulation of initiation in the small upstream open reading frame contributes to translation reg-ulation. Modulation of translation, and more gen-erally, efficiency in recognising an AUG context, might occur through mobilization of trans-acting factors as evidenced by McBratney and Sarnow [42].
Acknowledgements
This work was supported by grants from the Interuniversity Poles of Attraction Program-Bel-gian State, Prime Minister’s Office for Scientific, Technical and Cultural Affairs, the European Community’s BIOTECH program and the Belgian Fund for Scientific Research. We thank Dr J.-F. Briat (Montpellier) for providing the maize BMS suspension cells and Dr J.-L. Fourre´ (Louvain-la-Neuve) for providing the Norway spruce embryo-genic cells. We also thank Dr J.-F. Rees (Louvain-la-Neuve) and Dr J. Jacquemin (Gem-bloux) for providing use of luminometer and bi-olistic instruments, respectively.
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