Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Cellular differentiation wikipedia , lookup
List of types of proteins wikipedia , lookup
Cell nucleus wikipedia , lookup
Histone acetylation and deacetylation wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Transcription factor wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Eukaryotic transcription wikipedia , lookup
IN VITRO TRANSCRIPTION SUGIURA Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997. 48:383–98 Copyright © 1997 by Annual Reviews Inc. All rights reserved PLANT IN VITRO TRANSCRIPTION SYSTEMS Masahiro Sugiura Center for Gene Research, Nagoya University, Nagoya, 464-01, Japan KEY WORDS: plant genes, RNA polymerase I, Pol II, Pol III ABSTRACT In vitro transcription systems provide a powerful tool for detailed analysis of transcription reactions including initiation, elongation, and termination. Despite problems inherent to plant cells, efforts have been made to develop plant in vitro transcription systems in the past decade. These efforts have finally culminated in the development of reliable in vitro systems from suspension-cell cultures of both monocot and dicot plants. These systems can be useful in elucidating the specific mechanisms involved in the process of plant transcription and thus can potentially open a new era of transcription studies in plants. CONTENTS INTRODUCTION..................................................................................................................... PREPARATION OF IN VITRO TRANSCRIPTION SYSTEMS ........................................... Assay Methods ..................................................................................................................... Plant Materials .................................................................................................................... Preparation of Extracts ....................................................................................................... RNA POLYMERASE II-DEPENDENT TRANSCRIPTION.................................................. Extracts Prepared from Wheat Germ.................................................................................. Wheat Germ Chromatin Extract.......................................................................................... Cultured Cell Extracts ......................................................................................................... Evacuolated Protoplast Extracts......................................................................................... Convenient In Vitro Systems................................................................................................ POL I- AND POL III-DEPENDENT TRANSCRIPTION....................................................... Pol I Transcription .............................................................................................................. Pol III Transcription............................................................................................................ PROBLEMS AND PROSPECTS ............................................................................................. 384 385 385 387 387 388 388 389 390 392 392 394 394 394 395 383 1040-2519/97/0601-0383$08.00 384 SUGIURA INTRODUCTION The growth and development of plants and animals depend on the concerted expression of genes. Transcription is the essential regulatory step for the expression of nuclear genes encoding not only mRNAs but also stable RNAs, and this in turn involves a complex set of nucleic acid–protein and protein-protein interactions. Our current understanding of the fundamental and intricate mechanisms involved in the process and regulation of eukaryotic transcription owes a great deal to the in vitro systems prepared from animal and yeast cells. In vitro systems provide the most significant tool for analyzing individual steps in transcription, that is, initiation, elongation, and termination. Furthermore, in vitro functional assays allow the biochemical identification or isolation of factors involved in each step. These systems also facilitate screening of functionally significant motifs through the analysis of many deletion and/or scanning-substitution mutations. This would practically be impossible using transgenic plants. Furthermore, interaction of these motifs with trans-acting factors, whose availability might in turn depend on the internal and/or external cues, can also be studied using specific in vitro systems. These systems can be useful for studying the role of protein-protein interaction and the possible involvement of low–molecular mass substances in transcription. Therefore, the availability of versatile in vitro transcription systems from plant cells has long been awaited (32, 37, 47). To date, delineation of molecular aspects of transcription has mainly depended on time- and labor-consuming methods involving transgenic plants such as tobacco. In vitro transcription of several plant genes was also attempted using heterologous systems derived, for example, from HeLa cells and yeast cells. Such heterologous systems could faithfully initiate transcription from several plant genes, including those encoding maize zein (5, 6, 26), pea legumin (12), and cauliflower mosaic virus (CaMV) DNA (19). Heterologous systems could also be used to analyze basic processes of transcription and of similarity of promoter elements and protein factors between plants and animals, but they would not elucidate processes unique to plant cells. Therefore, no further advancements were made using these systems. Recent attempts were made to develop RNA polymerase II–dependent in vitro transcription systems derived from several plant species. However, these systems were either inefficient or of limited use. Finally, a long-awaited technical advance was achieved with the development of convenient, versatile, and reliable in vitro transcription systems from dicotyledonous (tobacco) and monocotyledonous (rice) plants (13, 48). IN VITRO TRANSCRIPTION 385 PREPARATION OF IN VITRO TRANSCRIPTION SYSTEMS Assay Methods In addition to the quality of the extract, the success of any in vitro system depends largely on the sensitivity of the assay method. A good assay method can selectively distinguish de novo synthesized RNAs (from exogenously added template DNA) from the extensive background consisting of endogenous transcripts from cell extracts, transcripts resulting from endogenous DNA incompletely removed during the preparation of extracts, RNA caused by nonspecific initiation and termination on the added template DNA, and products with nonspecific incorporation of a labeled substrate by side reactions. To achieve an acceptable level of sensitivity while minimizing the effect of these nonspecific contaminants, several assay methods have been tested. In vitro reactions are generally carried out using linear DNA templates, and transcription is allowed to proceed up to the end of the fragment. Specific initiation sites can be calculated from the size of a transcript(s) using polyacrylamide gel electrophoresis (PAGE). This assay is simple but requires higher activities of transcription elongation; otherwise, premature termination can cause unacceptably high levels of background. One way to enhance the detection of this procedure is the use of poly (dA) tail at the end of the template and subsequent selection of de novo full-length in vitro transcripts using oligo (dT) columns (30). Lower sensitivities are possible with this method because of the limitation of using linear templates, which are less active than circular templates (33, 48). Limited success in the past might result from the use of linear DNA templates in this assay. A similar procedure with circular DNA templates encoding small stable RNAs can facilitate the analysis of the transcription process as a whole, including initiation, elongation, and termination. RUN-OFF ASSAY Soon after the transcription reaction mixture is assembled—consisting of an extract, template DNA, and labeled nucleoside triphosphates, or NTPs, in a transcription buffer—initiation complexes form and synthesize short oligoribonucleotides. The reaction is stopped by addition of Sarkosyl, which removes most proteins from the DNA but leaves the stable transcription complexes intact. The amount of labeled oligoribonucleotides can be subsequently estimated by PAGE. Inefficiency of in vitro systems often results from a block in the elongation of RNA chains. This method can be used even when specific transcripts are not detected by other assays and can lead to the tentative identification of transcription initiation factors (1). TRANSCRIPTION COMPLEX ASSAY 386 SUGIURA S1 MAPPING AND RNASE PROTECTION ASSAYS Both of these methods work on the principle of specific hybridization of de novo transcripts to either labeled sense DNA strand or antisense RNA followed by nuclease digestion of unhybridized nucleic acid molecules. Thereafter, the site of transcription initiation is estimated by analyzing the size of nuclease-protected DNA/RNA fragments using PAGE. These methods are extremely sensitive, but they also detect signals from endogenous RNAs, transcripts resulting from endogenous DNA, and premature termination of in vitro reaction, with equally high sensitivity. Hence, this often results in exceptionally high backgrounds. Multiple sizes of product could indicate multiple initiation sites or multiple premature termination sites. Furthermore, sites outside the region of the probe cannot be detected. The template used in this assay contains a promoter region followed by a defined length of DNA (∼100 bp) lacking G residues on the sense strand in a plasmid vector. When the reaction is carried out in the absence of GTP or in the presence of RNase T1, RNA synthesis terminates at the first G in the plasmid sequence. Specific initiation at the promoter is determined by analyzing the size of transcripts. This assay is fast and quantitative and provides a more enzymological approach to the analysis of specific transcription initiation, e.g. purification of transcription factors (31). G-FREE SEQUENCE SYSTEM In this procedure, the in vitro products are used as templates for the synthesis of cDNAs (extended products) using reverse transcriptase (RT) and a (5′32P)-labeled primer complementary to a portion of template DNA used (23). Specific transcripts can be detected on gel by expected size for accurate initiation (from the 5′ end of primers to in vivo initiation sites). Because this assay uses only those in vitro transcripts that have elongated farther than the position of oligonucleotide primer, there can be a considerable reduction of background signals resulting from premature termination of an in vitro reaction. The use of primers complementary to a reporter gene sequence or a vector sequence (a nonplant DNA sequence) reduces background from nonspecific transcription from endogenous DNA or RNAs. This assay together with DNA sequence ladders resolves transcriptional initiation sites at the nucleotide level. Although the method is lengthier, it prevents artifactual results sometimes observed even when the G-free sequence system is used (14). One technical problem is that premature cessation of RT action results in multiple products; some plant genes are transcribed from several authentic sites but usually multiple-size fragments are an artifact. PRIMER EXTENSION ASSAY IN VITRO TRANSCRIPTION 387 Plant Materials The preparation of high-quality in vitro systems critically depends on the quality of starting material. For development of plant in vitro transcription systems, two main sources have been extensively exploited: wheat germ and suspension-cell cultures. Wheat germ has been widely used for the preparation of in vitro translation extracts that have also been reported to support pre-tRNA processing and splicing in vitro (35). It is the only plant source whose RNA polymerases have been extensively purified and characterized (21, 22). One of the reasons it is the material of choice is relatively low levels of nuclease and protease activities. However, it is not always easy to obtain good batches of wheat germ suitable for in vitro systems (42). Wheat germ is essentially a dormant material and may be deficient or inactivated with regard to factors required for general transcription (30). It is known to contain several inhibitors of the transcription that need to be removed before active systems can be developed (16, 18, 33). Extracts of isolated wheat shoot nuclei also contain inhibitors for transcription in a HeLa cell extract (20). WHEAT GERM Rapidly growing established cell cultures provide a continuous source of homogenous cells with defined growth characteristics. Suspension cultures of tobacco, rice, wheat, soybean, and parsley have been used for development of in vitro transcription systems. Because these cells contain large vacuoles, it is necessary to remove vacuole constituents from in vitro systems, e.g. by isolating nuclei or by fractionating whole cell extracts. Because these undifferentiated cells are probably devoid of any cell type–specific factors they are suitable for preparing “basal” in vitro transcription systems. SUSPENSION-CELL CULTURES Preparation of Extracts Plant cells generally contain high levels of proteases, nucleases, and other substances that may cause inactivation of transcription machinery during the preparation of extracts. Therefore, protease and phosphatase inhibitors and other protecting reagents are routinely added in extraction buffers. Moreover, the time of extract preparation should be minimized to reduce possible damage to the active constituents involved in transcription. Furthermore, plant cells are low in protein concentration compared with animal cells; therefore, a minimal volume of extraction buffers should be used to maintain higher protein concentrations. Nuclei, the site of transcription, are obviously the best choice for 388 SUGIURA extracts preparation; nevertheless, whole cells (or tissues) have also been used because they are easier to handle. Cells are disrupted mechanically by a Waring blender or a Bead Beater so that a large amount of starting material can be processed. Crude extracts containing endogenous DNA are generally inactive and require further fractionation, e.g. by column chromatography or ammonium sulfate precipitation. These treatments remove hazardous compounds as well as endogenous nucleic acids. WHOLE CELL EXTRACTS NUCLEAR EXTRACTS Using isolated nuclei as starting material avoids the potential hazard of cytoplasm (mainly vacuoles) and contamination of transcription activities from other organelles. For the isolation of intact nuclei, cells are gently disrupted, a step that is best achieved by osmotic lysis of protoplasts. Ficoll is added for adequate osmotic pressure to prevent the loss of nuclear contents by diffusion during preparation of nuclei. This is followed by extraction of nuclear proteins by high salt concentrations (30). Endogenous DNA is removed by ultracentrifugation. It is rather difficult to handle a large amount of cells using this procedure because of practical limitations. RNA POLYMERASE II-DEPENDENT TRANSCRIPTION RNA polymerase II (Pol II) is responsible for transcription of mRNAs and a class of U snRNAs. Purified Pol II recognizes and accurately transcribes mRNA promoters only when supplemented with additional factors present in crude cellular and nuclear extracts. In plant genes, most of the work has been devoted to the process of transcription initiation of mRNA genes, whose expression is differentially regulated by developmental and by external cues. Many cis-elements have been defined by transgenic assays, and a number of proteins interacting with DNA motifs have been isolated by affinity methods. There are a few studies elucidating detailed biochemical processes of transcriptional initiation, such as functional interaction between the cis-element and the DNA-binding protein. The analysis of these problems would be considerably facilitated by the use of in vitro transcription systems specific to plants. Extracts Prepared from Wheat Germ Initial attempts to develop in vitro Pol II–dependent transcription systems were made using wheat germ (1, 45). Ackerman et al (1) prepared extracts from IN VITRO TRANSCRIPTION 389 wheat germ whole cells, nuclei, and cytosols, and found that these extracts, especially a nuclear extract, support the formation of initiation complexes on externally added plant promoters. Although these extracts were not active in the Pol II run-off assay, some factors necessary for transcription initiation could be isolated by this assay method. This extract did not contain apparent RNase or DNase but inhibited transcription when added to a HeLa cell in vitro transcription system. The nuclear extract preparations were then fractionated, and inhibitors were removed by using a HeLa system for assays. A reconstituted system thus obtained selectively transcribed plant genes but produced an extremely low level of full-sized transcripts because of the apparent block in elongation at 20–30 nucleotides (nt) (16). A wheat protein fraction (termed KB) that substitutes for the HeLa TFIIA was isolated by substituting HeLa fractions with those of wheat using a HeLa in vitro system (7). The wheat TFIIA homologue was a single polypeptide of ∼35 kDa. The CaMV 35S promoter directed accurate and efficient transcription in the wheat/HeLa as well as a pure HeLa component assay. Therefore, the wheat TFIIA seems to convert Pol II to a more processive enzyme, which suggests that TFIIA functions during elongation and is also necessary for initiation complex assembly (10). Furthermore, wheat and HeLa TATA-binding proteins were enzymatically similar (S Ackerman, personal communication). This was also the case for a TATA-binding protein fractionated from Arabidopsis (27). These observations suggest that basic mechanisms of transcription are highly conserved among eukaryotes. Using the HeLa system, the minimal promoter of the CaMV 35S gene could be defined as −35 through TATA and the initiator sequence to +5 (34). Wheat Germ Chromatin Extract Yamazaki & Imamoto (45) prepared a soluble chromatin fraction from wheat germ crude extract by Polymer P fractionation followed by ammonium sulfate precipitation that was found to direct accurate transcription initiation from exogenously added transcript 7 gene (TC7) promoter of the T-DNA region of Ti plasmid, as measured by run-off and primer extension assays. The run-off transcript was estimated to amount to ∼80% of the total RNA synthesized, and its synthesis was completely inhibited by low concentrations of α-amanitin, a potent inhibitor for Pol II. This was the first report of accurate in vitro transcription of a plant-related gene. Using deletion experiments, they further suggested that the TATA-box is an important determinant of accurate initiation, and the region between +181 to +242 bp, in the middle of the coding region, is required for efficient initiation and elongation of TC7 transcription 390 SUGIURA in vitro. A striking characteristic of the system was that accurate transcription initiation was most active in the presence of 0.5-1 mM of Mn2+; far less activity was obtained using Mg2+ (44). The chromatin extract also supported accurate transcription initiation of a circular DNA containing the CaMV 35S promoter (44). Using this in vitro system with the 35S promoter containing the TATA-box and the upstream activation sequence, as-1, the tobacco DNAbinding protein TGA1a for as-1 was reported to stimulate transcription threeto fivefold through increasing the number of active preinitiation complexes (46). TGA1a can also function as a transcription activator in a HeLa system with the 35S promoter (24), again suggesting high conservation of basic processes of transcription in eukaryotes. Using the chromatin extracts with the G-free sequence assay, Schweizer & Mösinger (33) examined the sequence requirements for faithful and efficient transcription initiation of a series of chimeric promoter constructs from several plant genes. They found that the chromatin extract transcribes the parsley chalcone synthase promoter in an initiator sequence- (encompassing the transcriptional start site, positions −7 to +13) dependent manner, but not in a TATA-dependent manner. Their result was not in agreement with the earlier report on TATA-box–dependent in vitro transcription from a linear TC7 promoter (45) and a circular CaMV 35S DNA (44). In their analysis, requirement for circular DNA was obligatory, and presence of 2 mM Mg2+ was preferred over 1 mM Mn2+. Moreover, the in vitro transcription initiation site of the parsley pathogenesis-related protein 2 promoter was different from the in vivo site [24 nt upstream from the in vivo site (33)]. These observations suggested that the wheat germ chromatin extract represented only a partly functional in vitro system, and in vitro systems from wheat germ would need further refinement before they could be used for transcription analysis using genuine plant promoters. Cultured Cell Extracts Efforts were also made to develop in vitro transcription systems from several plant cultured cells. Cooke & Penon reported that although a whole cell extract is transcriptionally inactive, an in vitro system derived from tobacco-cultured cells only after a single chromatographic separation can direct selective transcription from the CaMV 19S promoter (8, 9). Using this system, transcription from the 35S promoter led to the accumulation of short RNAs, although the 19S promoter was suggested to be weaker than the most widely used 35S promoter (19). IN VITRO TRANSCRIPTION 391 Roberts & Okita (30) presented a simple method for the preparation of nuclear extracts from cultured cells of rice, wheat, or tobacco. These extracts were shown to initiate transcription from the wheat gliadin and CaMV 35S promoters. They used protoplasts to prepare the nuclear extracts from cultured cells. In their procedures, high concentration of Ficoll was used during nuclear isolation, nuclear proteins were recovered using ammonium sulfate, and oligo (dT) selection of run-off transcripts was employed for their assay to reduce nonspecific transcripts. A major problem in using cultured cell extracts is the high background levels, which hinder the identification and quantification of desired exogenous template-dependent transcription products. To avoid this problem, Arias et al (3) used immobilized DNA templates. Transcription complexes were assembled on the soybean chalcone synthase 15 promoter (CHS 15)–containing templates coupled to agarose beads using homologous whole cell and nuclear extracts from soybean-cultured cells. These beads were then washed to remove unbound materials and incubated with labeled substrates to allow transcription. While increasing the recovery of exogenous DNA-dependent transcripts, the washing of immobilized transcription complexes considerably reduced the background. This homologous in vitro system directed accurate and efficient transcription initiation from the CHS15 promoter. By using this system, it was shown that trans factors that bind to G-box (CACGTG, −74 to −69) and H-box (CCTACC, −61 to −56 and −121 to −126) cis-elements, respectively, greatly contribute to the transcription of the CHS15 promoter in vitro, and that both cis-element/trans-factor interactions in combination are required for maximal activity. Authentic transcription from the CHS15 promoter was also observed with whole cell extracts from bean-, tobacco-, and rice-cultured cells, and the soybean whole cell extract transcribed several other immobilized promoters. Although it requires additional steps (e.g. immobilization and washing) and confines transcription to a single cycle per assay, the system is useful for analysis of transcriptional initiation of plant promoters. Yamaguchi et al (42) also reported an in vitro system from isolated nuclei of tobacco-cultured BY-2 cells after protoplast formation. Their system directs accurate transcription initiation from the TC7 promoter by using G-free sequence assay. Characteristically, the assay was again found to be most accurate and active only in the presence of 1 mM Mn2+. The requirement of Mn2+, therefore, seems to be unique to the TC7 or a limited class of plant promoters. However, Mn2+ is known to alter the specificity of templates and substrates in DNA and RNA polymerase reactions (11). 392 SUGIURA Evacuolated Protoplast Extracts Plant cells have large vacuoles; when ruptured they dilute the extracts and contain many compounds that may damage transcription components. Frohnmeyer et al (17) reported a light-responsive in vitro transcription system from evacuolated protoplasts of parsley-cultured cells. They further showed that light-treated lysates preferentially enhance the transcription activity from a transformed parsley chalcone synthase gene compared with dark-treated lysates. Similar conditions, however, had no enhancement effect on the constitutively transcribed transformed CaMV 35S promoter. Although their system cannot be used for externally added templates in the reaction mixtures, it remarkably retains the light responsiveness for the CHS gene, indicating the maintenance of a largely intact signal transduction chain between photoreceptor(s) and the promoter. Thus if exploited, it can prove to be a valuable tool for elucidating signaling mechanisms, at least for light response. Convenient In Vitro Systems The in vitro transcription systems described above were either of limited use or needed special skill or laborious procedures. Recently, a long-awaited technical advance has been achieved in the development of convenient and versatile in vitro systems by two groups (37, 47). Zhu et al (48) have developed a convenient in vitro transcription system using rice and tobacco whole cell extracts and circular DNA templates. Using a procedure based on that reported by Arias et al (3), they prepared extracts from suspension cultures in the exponential to early stationary phases of growth under carefully controlled culture conditions. Breakage of cells was done using a Bead Beater homogenizer, and the homogenate was fractionated using ammonium sulfate. The transcription reactions were optimized using templates containing homologous promoters either from the rice phenylalanine ammonia-lyase (PAL) gene or from the tobacco sesquiterpene cyclase gene. Accurate initiation of transcription, using circular but not linear templates, was unambiguously detected by primer extension assays. The optimized rice in vitro system supports three to four cycles of transcription on each transcriptionally competent template per assay, enough for a wide range of applications for analyzing the Pol II–dependent transcription. Moreover, these extracts can be stored more than one year at −80°C without losing significant activity. Using this system, Zhu et al (49) pursued detailed dissection of the functional architecture of plant minimal promoters using the rice extracts with many mutagenized promoters from the rice PAL gene. This proved for the first time that the TATTTAA sequence (positions −35 to −28) is an authentic IN VITRO TRANSCRIPTION 393 TATA-box essential for Pol II–dependent transcription. Moreover, the −1 and +1 sites of the initiator sequence and the spacing between the TATA box and initiation site were necessary for the correct placement of the initiation site. Their key findings were confirmed by in vivo experiments using a homologous system—a rice gene, rice extracts, and rice plants. A rice basic chitinase gene and a rice tungro bacilliform virus promoter were also accurately transcribed (41, 47). Furthermore, several DNA-binding proteins that are possibly involved in the regulation of rice gene transcription have been isolated from the rice extract (47; Q Zhu, personal communication). We have also developed a basal in vitro transcription system from rapidly grown, nongreen cultured BY-2 cells of tobacco (13, 14). Intact nuclei were isolated from protoplasts and disrupted using a high concentration of salt followed by ultracentrifugation to remove endogenous DNA. The in vitro reaction was optimized using a circular DNA template containing the tobacco β-1,3-glucanase promoter and primer extension assay. The system supported accurate transcription initiation not only from tobacco β-1,3-glucanase gene but also the CaMV 35S promoter, the adenovirus 2 major late promoter, and the simian virus 40 early major promoter. The tobacco nuclear extract supported ∼1.5 cycles of transcription on each transcriptionally competent template per assay. Nuclear genes encoding components of the photosynthesis apparatus are actively transcribed in green leaves under illumination but are poorly expressed in dark-grown plants and other nonphotosynthetic organs. The tobacco in vitro system was then used to transcribe a tomato (close to tobacco) gene encoding the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS3C) whose expression is tissue specific and light dependent. As expected because BY-2 cells are undifferentiated and contain nonphotosynthetic plastids, the tomato promoter was inactive in the BY-2 in vitro system, whereas accurate transcription of this promoter was observed by supplementing the BY-2 system with a nuclear extract from light-grown tomato seedlings. By using the activated in vitro system, the functional elements of the rbcS3C promoter were analyzed. A sequence 351 bp upstream from the transcription initiation site was essential for transcription, and the region between −351 to −441 bp enhances transcription. The basal/activated BY-2 system may be useful for analyzing a wide range of Pol II–dependent transcription reactions and for identifying specific signals from differentiated tissues or organelles, which modulate transcription activity. This system also supports Pol I– and Pol III–dependent transcription, making it the most versatile system so far. 394 SUGIURA POL I– AND POL III–DEPENDENT TRANSCRIPTION Pol I Transcription Pol I transcribes tandemly repeated rRNA gene (rDNA) clusters consisting of 17S rDNA, 5.8S rDNA, and 25S rDNA. Moreover, Pol I–dependent transcription is known to be species specific, at least in mammalian cells. Yamashita et al (43) recently reported an in vitro system for transcription of the Vicia faba rDNA. The soluble whole cell extract prepared from V. faba embryonic axes initiated transcription of a linear template containing a V. faba rDNA promoter in the presence of α-amanitin, which inhibits Pol II (at a low concentration) and Pol III (at a high concentration) but not Pol I. In addition, the major transcript species was determined by run-off and primer extension analyses to initiate at the same position previously determined by S1 analysis in vivo. Using the tobacco in vitro system, we also showed transcription from a circular tobacco rDNA template as determined by primer extension (13, 15). The transcription initiation site was found at an A, which corresponds to that lying within the consensus sequence surrounding plant pre-rRNA initiation (28). Because this transcription was resistant to a high concentration of αamanitin, the in vitro system most likely supports accurate initiation of tobacco rDNA transcription by Pol I. The in vivo transcription initiation site of tobacco rDNA has not been determined because of rapid processing of pre-rRNA (K Yakura, personal communication). The in vitro assay can help in overcoming this difficulty. The system, however, did not support transcription of V. faba rDNA, indicating that plant Pol I–dependent rDNA transcription is also species specific (15). Pol III Transcription Pol III is responsible for transcription of tRNAs, 5S rRNA, U6 snRNA, and 7SL RNA. Plant U3 snRNA genes were transcribed by Pol III and not by Pol II as in vertebrates or lower eukaryotes (25). A gene encoding the major cytoplasmic tRNATyr from Nicotiana rustica contains a 13-bp intron and was transcribed efficiently by Pol III in a HeLa nuclear extract (40). The pre-tRNA was subsequently processed and spliced into its mature size tRNATyr in the HeLa extract. Wheat germ extracts were then shown to accurately process, splice, and modify the pre-tRNATyr transcribed in HeLa extracts (35). Human and yeast cell extracts were also shown to transcribe several other plant tRNA genes (2, 4, 29, 36, 38, 39). We estimated Pol III activity to be ∼10% of the total RNA polymerase activities in the tobacco in vitro system (13). Therefore, in vitro transcription IN VITRO TRANSCRIPTION 395 from a circular DNA template containing an Arabidopsis U6 snRNA promoter was conducted, and the in vitro products were assayed by primer extension. Transcription initiated at the same site as in vivo, and it was resistant to a low concentration of α-amanitin, which inhibits Pol II, indicating that the tobacco in vitro system also supports accurate initiation of U6 transcription. We can improve the tobacco system with respect to its Pol III activity, which now allows us to carry out run-off assay with full-size genes. The improved system supported accurate transcription initiation and termination from Arabidopsis U3, U6, and 7SL RNA genes, and also from Arabidopsis tRNASer genes (Y Yukawa, unpublished data). In vitro transcripts from these Pol III–dependent genes are small (∼100 nt) and stable. Therefore we are now in a position to investigate the transcription process in its totality, i.e. initiation, elongation, and termination. PROBLEMS AND PROSPECTS Because of the lack of reliable plant in vitro transcription systems, our understanding of transcription processes and their regulation in plants has not been at par with that in animals and yeast. Hence, the gap has often been filled using the knowledge obtained from animal and yeast systems. This might not always be right; for example, plant U3 snRNA genes were transcribed by Pol II and not by Pol III, as in animals and yeast (25). Recently, in vitro transcription systems, which are potentially useful for studying a wide range of transcription processes, from suspension-cultured cells of rice and tobacco have been reported. Results obtained by in vitro experiments are often necessary to be confirmed by in vivo analysis, or vice versa. Rice and tobacco have been the best choices because these have also been favorites for in vivo studies regarding gene expression, including transgenic assays. Moreover, these plant cells can easily and economically be cultured in a large amount necessary for isolating transcription factors. The plant in vitro transcription system is an essential tool to understand plant-specific processes, such as light-response and chloroplast signals. For wide use, the in vitro system should be reproducible in other laboratories, and should be as simple and versatile as possible. Therefore, further improvement and optimization of existing plant in vitro systems are still necessary. Because of the complex nature of transcription (also splicing and translation), protocols for extract preparation are not as complete and elaborate as those of DNA sequencing or gene cloning. Therefore, personal experience is often necessary to obtain active extracts. Fractionation of extracts and reconstitution of subfractions or purified components are the next steps in understanding transcrip- 396 SUGIURA tion in plants. Nevertheless, plant in vitro transcription systems have presented encouraging possibilities for the analysis of biochemical processes of transcription from plant genes and may provide new insights into processes that are unique to plant cells. ACKNOWLEDGMENTS I thank Sanjay Kapoor for critical reading of the text. I am also grateful to M Sugita, Y Yukawa, K Yakura, S Ackerman, Q Zhu, K Yamazaki, H Beier, R Cooke, and P Schweizer for providing information. Visit the Annual Reviews home page at http://www.annurev.org. Literature Cited 1. Ackerman S, Flynn PA, Davis EA. 1987. Partial purification of plant transcription factors. I. Initiation. Plant Mol. Biol. 9: 147–58 2. Arends S, Kraus J, Beier H. 1996. The tRNATyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron-containing pre-tRNAs in wheat germ extract. FEBS Lett. 384:222–26 3. Arias JA, Dixon RA, Lamb CJ. 1993. Dissection of the functional architecture of a plant defense gene promoter using a homologous in vitro transcription initiation system. Plant Cell 5:485–96 4. Beier D, Beier H. 1992. Expression of variant nuclear Arabidopsis tRNASer genes and pre-tRNA maturation differ in HeLa, yeast and wheat germ extracts. Mol. Gen. Genet. 233:201–8 5. Boston RS, Goldsbrough PB, Larkins BA. 1985. Transcription of a zein gene in heterologous plant and animal systems. In Plant Genetics, ed. M Freeling, pp. 629–39. New York: Liss 6. Boston RS, Larkins BA. 1986. Specific transcription of a 15-kilodalton zein gene in HeLa cell extracts. Plant Mol. Biol. 7: 71–79 7. Burke C, Yu XB, Marchitelli L, Davis EA, Ackerman S. 1990. Transcription factor IIA of wheat and human function similarly with plant and animal viral promoters. Nucleic Acids Res. 18:3611–20 8. Cooke R, Penon P. 1990. In vitro transcription from cauliflower mosaic virus promot- 9. 10. 11. 12. 13. 14. 15. 16. ers by a cell-free extract from tobacco cells. Plant Mol. Biol. 14:391–405 Cooke R, Penon P. 1990. In vitro transcription of class II promoters in higher plants. Methods Mol. Biol. 49:271–89 de Mercoyrol L, Job C, Ackerman S, Job D. 1989. A wheat-germ nuclear fraction required for selective initiation in vitro confers processivity to wheat-germ RNA polymerase II. Plant Sci. 64:31–38 Dixon M, Webb EC. 1980. Enzyme biosynthesis. In Enzymes, pp. 570–621. London: Longman. 3rd ed. Evans IM, Bown D, Lycett GW, Croy RRD, Boulter D, Gatehouse JA. 1985. Transcription of a legumin gene from pea (Pisum sativum L.) in vitro. Planta 165:554–60 Fan H, Sugiura M. 1995. A plant basal in vitro system supporting accurate transcription of both RNA polymerase II- and IIIdependent genes: supplement of green leaf component(s) drives accurate transcription of a light-responsive rbcS gene. EMBO J. 14:1024–31 Fan H, Sugiura M. 1996. Basal and activated in vitro transcription in plants by RNA polymerase II and III. Methods Enzymol. 273:268–77 Fan H, Yakura K, Miyanishi M, Sugita M, Sugiura M. 1995. In vitro transcription of plant RNA polymerase I-dependent rRNA genes is species-specific. Plant J. 8:295–98 Flynn PA, Davis EA, Ackerman S. 1987. Partial purification of plant transcription factors. II. An in vitro transcription system is inefficient. Plant Mol. Biol. 9:159–69 IN VITRO TRANSCRIPTION 17. Frohnmeyer H, Hahlbrock K, Schäfer E. 1994. A light-responsive in vitro transcription system from evacuolated parsley protoplasts. Plant J. 5:437–49 18. Furter R, Hall BD. 1991. Substances in nuclear wheat germ extracts which interfere with polymerase III transcriptional activity in vitro. Plant Mol. Biol. 17:773– 85 19. Guilley H, Dudley RK, Jonard G, Balàzs E, Richards KE. 1982. Transcription of cauliflower mosaic virus DNA: detection of promoter sequences, and characterization of transcripts. Cell 30:763–73 20. Henfrey RD, Proudfoot LMF, Covey SN, Slater RJ. 1989. Identification of an inhibitor of transcription in extracts prepared from wheat shoot nuclei. Plant Sci. 64: 91–98 21. Jendrisak J. 1981. Purification and subunit structure of DNA- dependent RNA polymerase III from wheat germ. Plant Physiol. 67:438–44 22. Jendrisak JJ, Burgess RR. 1975. A new method for the large-scale purification of wheat germ DNA-dependent RNA polymerase II. Biochemistry 14:4639–44 23. Kadonaga JT. 1990. Assembly and disassembly of the Drosophila RNA polymerase II complex during transcription. J. Biol. Chem. 265:2624–31 24. Katagiri F, Yamazaki K, Horikoshi M, Roeder RG, Chua NH. 1990. A plant DNAbinding protein increases the number of active preinitiation complexes in a human in vitro transcription system. Genes Dev. 4:1899–909 25. Kiss T, Marshallsay C, Filipowicz W. 1991. Alteration of the RNApolymerase specificity of U3 snRNA genes during evolution and in vitro. Cell 65:517–26 26. Langridge P, Feix G. 1983. A zein gene of maize is transcribed from two widely separated promoter regions. Cell 34:1015–22 27. Mukumoto F, Hirose S, Imaseki H, Yamazaki K. 1993. DNA sequence requirement of a TATA element-binding protein from Arabidopsis for transcription in vitro. Plant Mol. Biol. 23:995–1003 28. Perry KL, Palukaitis P. 1990. Transcription of tomato ribosomal DNA and the organization of the intergenic spacer. Mol. Gen. Genet. 221:102–12 29. Reddy PS, Padayatty JD. 1988. Effects of 5’ flanking sequences and changes in the 5’ internal control region on the transcription of rice tRNAGlyGCC gene. Plant Mol. Biol. 11:575–83 30. Roberts MW, Okita TW. 1991. Accurate in vitro transcription of plant promoters with 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 397 nuclear extracts prepared from cultured plant cells. Plant Mol. Biol. 16:771–86 Sawadogo M, Roeder RG. 1985. Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay. Proc. Natl. Acad. Sci. USA 82:4394- 98 Schweizer P. 1994. In vitro transcription of plant nuclear genes. In Results and Problems in Cell Differentiation, ed. L Nover, 20:105–21. Berlin: Springer-Verlag Schweizer P, Mösinger E. 1994. Initiatordependent transcription in vitro by a wheat germ chromatin extract. Plant Mol. Biol. 25:115–30 Sif S, Cummings A, Davis EA, Ackerman S. 1993. Interaction of human transcription factors IIA and IID with the cauliflower mosaic virus 35S promoter. Mol. Biol. 12: 53–61 Stange N, Beier H. 1987. A cell-free plant extract for accurate pre tRNA processing, splicing and modification. EMBO J. 6: 2811-18 Stange N, Beier D, Beier H. 1991. Expression of nuclear tRNATyr genes from Arabidopsis thaliana in HeLa cell and wheat germ extracts. Plant Mol. Biol. 16:865– 75 Sugiura M. 1996. Plant in vitro transcription: the opening of a new era. Trends Plant Sci. 1:41 Teichmann T, Urban C, Beier H. 1994. The tRNASer-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and sequence analyses. Plant Mol. Biol. 24:889–901 Ulmasov B, Folk W. 1995. Analysis of the role of 5′ and 3′ flanking sequence elements upon in vivo expression of the plant tRNATrp genes. Plant Cell 7:1723–34 van Tol H, Stange N, Gross HJ, Beier H. 1987. A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways. EMBO J. 6:35–41 Xu Y, Zhu Q, Panbangred W, Shirasu K, Lamb C. 1996. Regulation, expression and function of a new basic chitinase gene in rice (Oryza sativa L.). Plant Mol. Biol. 30:387–401 Yamaguchi Y, Mukumoto F, Imaseki H, Yamazaki K. 1994. Preparation of an in vitro transcription system of plant origin, with methods and templates for assessing its fidelity. In Plant Molecular Biology Manual, ed. SB Gelvin, RA Schilperoot, pp. 1–15. Dordrecht: Kluwer. 2nd ed. Yamashita J, Nakajima T, Tanifuji S, Kato A. 1993. Accurate transcription initiation of 398 SUGIURA Vicia faba rDNA in a whole cell extract from embryonic axes. Plant J. 3:187–90 44. Yamazaki K, Chua NH, Imaseki H. 1990. Accurate transcription of plant genes in vitro using a wheat germ-chromatin extract. Plant Mol. Biol. Rep. 8:114–23 45. Yamazaki K, Imamoto F. 1987. Selective and accurate initiation of transcription at the T-DNA promoter in a soluble chromatin extract from wheat germ. Mol. Gen. Genet. 209:445–52 46. Yamazaki K, Katagiri F, Imaseki H, Chua NH. 1990. TGA1a, a tobacco DNA-binding protein, increases the rate of initiation in a plant in vitro transcription system. Proc. Natl. Acad. Sci. USA 87:7035–39 47. Zhu Q. 1996. RNA polymerase II-dependent plant in vitro transcription systems. Plant J. 10:185–88 48. Zhu Q, Chappell J, Hedrick SA, Lamb C. 1995. Accurate in vitro transcription from circularized plasmid templates by plant whole cell extracts. Plant J. 7:1021–30 49. Zhu Q, Dabi T, Lamb C. 1995. TATA box and initiator functions in the accurate transcription of a plant minimal promoter in vitro. Plant Cell 7:1681–89