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The Activation Process Ofarabidopsis Thaliana A1 Gene Encoding The The activation process ofArabidopsis thaliana A1 gene encoding the translation elongation factor EF-I is conserved among angiosperms Catherine Curie, Thierry Liboz, Marie-Hélène Montané, Dominique Rouan, Monique Axelos, Bernard Lescure To cite this version: Catherine Curie, Thierry Liboz, Marie-Hélène Montané, Dominique Rouan, Monique Axelos, et al.. The activation process ofArabidopsis thaliana A1 gene encoding the translation elongation factor EF- I is conserved among angiosperms. Plant Molecular Biology, Springer Verlag (Germany), 1992, 18, pp.1083-1089. 10.1007/BF00047711. hal-03081959 HAL Id: hal-03081959 https://hal.archives-ouvertes.fr/hal-03081959 Submitted on 18 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution - NonCommercial - NoDerivatives| 4.0 International License Plant Molecular Biology 18: 1083-1089, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium. 1083 The activation process ofArabidopsis thaliana A1 gene encoding the translation elongation factor EF-I is conserved among angiosperms Catherine Curie 1, Thierry Liboz 1, Marie-H61bne Montan62, Dominique Rouan 1, Mich+le Axelos l and Bernard Lescure 1, 1Laboratoire de Biologie MolOculaire des Relations Plantes-Microorganismes, CNRS-INRA, BP 27, 31326 Castanet-Tolosan Cedex, France (* author for correspondence); 2 Rh6ne Poulenc Secteur Agro, 14-20 rue Pierre Baizet, BP 9163, 69263 Lyon Cedex 09, France Received 28 June 1991; accepted in revised form 7 January 1992 Key words: translation elongation factor genes, promoter analysis, evolution Abstract In Arabidopsis thaliana, the activation process of the A1 EF-lc~ gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis- acting elements of the A1 promoter, or the deletion of an intron located within the 5' non-coding re- gion, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5' intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculenturn EF-1 ~ promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-I~ genes have been conserved among an- giosperms and provide interesting data on the functional structure of the TEF 1 box. Introduction In A. thaliana, EF-le is encoded by a small multigenic family of four members (A l-A4) which The elongation factor EF-le belongs to the are all actively transcribed [ 1, 19]. Using Arabi- housekeeping protein family and has been inten- dopsis protoplasts, transfection experiments have sively studied because of its central role in the shown that the A1 promoter mediates a transient protein synthesis process [23]. In spite of these expression higher than that obtained using the studies, except for Saccharornyces cerevisiae [15] cauliflower mosaic virus (CaMV) 35S promoter, and Arabidopsis thaliana [8], there are no pub- one of the strongest promoters known both in lished data concerning the characterization of cis- transient expression systems and stably trans- and trans-acting elements involved in the activa- formed plant cells [1]. This high level of ex- tion of EF-lc~ genes. pression appears to be conserved in transgenic 1084 Arabidopsis (unpublished data). Deletion analysis was used for the transfection of Z. mays proto- of A1 promoter has shown that several elements plasts [9]. In all cases, 20 #g of plasmid DNA, are involved in the transcription activation pro- carrying the indicated promoter configuration cess in transfected Arabidopsis cells [8]. The fused to the GUS reporter gene, were used to location of these elements within the A 1 promoter transfect 106 protoplasts. The GUS activity was is indicated in Fig. 1A. A DNA sequence located measured after 20 h by fluorometry [ 16]. upstream of position -289 relative to the tran- scription initiation site is required for a maximum activity. One cis-acting domain, the TEF 1 box, Gel retardation assays has been accurately mapped 100 bp upstream of transcription initiation site. This domain is the Crude nuclear extracts were prepared from target for trans-acting factors identified in nuclear young leaves of B. rapa, N. tabacum or Z. mays, extracts prepared from Arabidopsis leaves or cell using the procedure described for the prepara- suspension cultures. In addition, we have shown tion of A. thaliana nuclear extracts [8]. Binding that the A 1 gene contains an intron, located within assays were done in a volume of 30 #1 containing the 5' non-coding region. Evidence has been 10 mM TRIS-HC1 pH 8, 50 mM NaC1, 7 mM obtained that this intron is essential to the ex- 2-mercaptoethanol, 10 70 glycerol, 2 #g of pression of the GUS reporter gene used in the poly(dI-dC), 1000-5000 cpm (0.1-0.5 ng) of transient expression experiments. [8]. 32p-labelled probe and 2-5 #g of nuclear proteins. Here, we report results showing that mutations After 20 min at 25 ° C, the free and bound DNA of the A1 promoter similarly affect the expression were separated on 570 polyacrylamide gels in in dicot or monocot protoplasts and that factors 0.5x TBE. interacting with the TEF 1 box are present in nu- clear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Zea mays. More- Results over, we show that a related DNA sequence found at approximately the same location within the Le In order to determine whether the activity of EF-1 Lycopersicon esculentum EF-lc~ promoter cis elements involved in the activation of the [25], specifically interacts with the same trans- A. thaliana EF-I~ A1 promoter was conserved acting factors. These data have allowed us to de- among angiosperms, characteristic promoter mu- fine by mutagenesis, two important domains tations were tested in N. tabacum and Z. mays within the TEF 1 box. protoplasts. Figure 1A shows a schematic repre- sentation of the A1 promoter, with the location of the relevant cis elements [8]. The GUS activities Material and methods given in Fig. 1B are expressed relative to the full- length promoter (x-TEF-TATA-IVS configura- Transient gene expression assays tion: 2300 bp upstream of the AUG translation initiation codon fused to the GUS reporter gene). The plasmid constructions used to transfect the As in A. thaliana, in transfected Z. mays proto- protoplasts have been already described [ 8 ]. The plasts the full-length A 1 promoter appears to drive protoplasts were prepared from cell suspension a GUS activity higher than that obtained using cultures of A. thaliana [ 1 ] and of Z. mays (49S 1 the CaMV 35S promoter (CaMV 35S configura- embryogenic cell line developed by T. Hardy, tion). A 5' deletion down to the position -111 Rh6ne Poulenc Agro), or from N. tabacum leaves relative to the transcription initiation site (TEF- [ 7 ]. For A. thaliana and N. tabacum, transfections TATA-IVS configuration), led to about a two- were performed according to the Ca(NO3)z - fold decrease in the GUS activity. This moderate PEG procedure [22]. The electroporation method effect, which depends on a cis-acting element 1085 D TATA // -t620 -289 ~/+56 +657 I rr ATG " -106 /2 -82 +678 7--~--I TEF1 I 5'IVS I ® Promoter configuration A. thaliana N. tabacum Z. mays Fig. 2. Identification by gel retardation of trans-acting factors interacting with the TEF1 box. The probes used extend from X-TEF-TATA-IVS 100 100 100 the 5' position - 100 or - 111 to the position + 46 relative to TEF-TATA-IVS 40+/-5 60+•-2 51+/-8 the transcription initiation site. The origin of nuclear extracts TATA-IVS 2,5+/-0,2 4+/-1 3+•-2 used is indicated. HFT and H450 correspond to C1 and C2 TEF-TATA 3+/-0,2 ND 2+/-1 complexes previously described [8] (see the text for details). CaMV 35S 42+/-5 ND 21+/-4 Fig. 1. A. Schematic representation showing the location of transcription initiation site and that these two EF-lc~ A1 promoter elements involved in the activation of complexes disappear using a probe deleted down expression [8]. The positions are relative to the transcription to position -100 [8]. Therefore, the same DNA initiation site. See the text for details. B. Transient expression fragments ( - 111/+ 46 and - 100/+ 46) were used in A. thaliana, N. tabacum and Z. mays protoplasts. The na- as probes to test for the presence of ture of the promoter configuration indicated is detailed in the trans-acting text. The relative GUS activities, normalized to the full length factors specifically interacting with the TEF 1 promoter (1007o), are the mean of data from at least three independent transfections. After 20 h, the GUS activity mea- sured for the full length promoter was 120, 6 or 30 nmol/min per mg protein for A. thaliana, N. tabacum or Z. mays proto- plasts respectively. ND, not determined.
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