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IN VITRO TRANSCRIPTION
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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 .............................................................................................
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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).
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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
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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
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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
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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
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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
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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).
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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).
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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
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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.
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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
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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-
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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.
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