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J Biol Chem, Vol. 275, Issue 13, 9215-9221, March 31, 2000
From the Laboratoire de Génétique Moléculaire des Plantes, "Plastes et Differenciation Cellulaire," Université Joseph Fourier and CNRS, B.P. 53X, F-38041 Grenoble, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Three different cDNAs coding for putative
plant plastid sigma70-type transcription initiation
factors have recently been cloned and sequenced from Arabidopsis
thaliana. We have analyzed the evolutionary conservation of
function(s) of the N-terminal and C-terminal halves of these three
sigma factors by in vitro transcription studies using
heterologous transcription systems and by complementation assays using
Escherichia coli thermosensitive rpoD mutants.
Our results indicate differences and similarities of the three plant factors and their prokaryotic ancestors. The functions of the N-terminal parts of the plant sigma factors are considerably different from the function of the N-terminal part of the principal
sigma70 factor of E. coli. On the other hand,
the C-terminal parts have kept at least two characteristics when
compared with their prokaryotic ancestors: 1) they can distinguish
between different promoter structures, and 2) one of them is capable of
fully complementing E. coli rpoD mutants, i.e.
recognizing all essential E. coli promoters that are used
by the E. coli principal sigma70 factor. This
shows for the first time in vivo a strong evolutionary conservation of cis- and trans-acting elements between the prokaryotic and the plant plastid transcriptional machinery.
The higher plant plastid genome is transcribed by two different
transcriptional systems (reviewed in Ref. 1). One of the two systems is
phage-like and its evolutionary origin is still unclear (2, 3). The
other system is of the prokaryotic type revealing the cyanobacterial
origin of present-day chloroplasts (4-6). All polypeptides that are
necessary to build up the core enzyme of the prokaryotic-type plastid
RNA polymerase are still encoded in the plastid genome (7, 1). However,
genes corresponding to sigma-like transcription initiation factors that
are indispensable for the activity of this type of enzyme (reviewed in
Ref. 8) are not found on the plastid genomes (7, 1). These genes have
been transferred to the nucleus during evolution (9).
The existence of more than one sigma-like transcription factor in
higher plant plastids had been suggested several years ago on the basis
of biochemical approaches for spinach and mustard (10-12). However,
the corresponding genes have not been cloned. Only recently, six
different cDNAs showing strong sequence similarity with genes
coding for prokaryotic-like sigma70-type initiation factors
have been cloned and sequenced from Arabidopsis thaliana
(accession numbers: SIG1, dbj: AB004820 and emb: Y15362; SIG2, dbj:
AB004821 and emb: Y14567; SIG3, dbj: AB004822; SIG4, gb: AF101075;
SIG5, emb: Y18550; and SIG6, emb: AJ250812). For three of them, it has
been shown that the corresponding proteins are transported into the
chloroplasts (9, 14), but the function of these putative transcription
factors in the regulation of plastid gene expression is not clear. In
particular, it is not clear why several sigma70-type
factors have been conserved during evolution, despite an important
reduction of (plastid) genome size.
Prokaryotic sigma factors can be classified into two families. Members
of the first family are similar to the Escherichia coli
sigma70 factor, whereas members of the other family are
similar to the E. coli sigma54 factor. The
sigma70 family of transcription factors can be further
subdivided into two or three groups. One primary sigma factor is
required to ensure that "housekeeping functions" are performed, and
alternative sigma factors direct transcriptional responses to changing
environmental conditions (8). Although both groups of
sigma70 factors are similar with respect to their amino
acid sequence, they recognize different sequences at the two promoter
elements localized -10 and -35 base pairs upstream of the
transcription start sites (reviewed in 8 and 15).
In the present study, we attempted to obtain information about the
evolutionary conservation of components of the transcriptional machinery between prokaryotes and higher plant plastids. With this aim,
we analyzed differences of the three plant sigma factors with respect
to promoter recognition by in vitro transcription, and
we determined the plant analogue to the primary sigma factor of
E. coli by complementation of E. coli
rpoD(ts) mutants. For the first time, we show in
vivo complementation of a protein of the E. coli
transcriptional machinery by a protein of the plastid transcriptional machinery.
Isolation of Clones Coding for A. thaliana Plastid Sigma
Factors--
A. thaliana cDNA and/or genomic sequences,
similar to prokaryotic sigma factors, have been searched for in
currently available data banks. Six such sequences have been found.
Four of them have been detected as partial cDNA sequences in the
data base expressed sequence tag (dbEST), and two of them have been
identified in the genomic sequences resulting from the
Arabidopsis Genomic Initiative. The corresponding
full-length cDNA clones were isolated from an A. thaliana Matchmaker cDNA library
(CLONTECH) either by screening with the
corresponding A. thaliana EST clones or by direct
PCR1 amplification (accession
numbers: SIG1, Y15362; SIG2, Y14567; SIG5, Y18550; and SIG6,
AJ250812).
Overproduction in E. coli and Purification of Recombinant
Proteins--
Sequences corresponding to full-length and truncated
forms of SIG1, SIG2, and SIG3 polypeptides were PCR-amplified using the following primers at the 5' ends: SIG1-KLR,
5'-CGCGGATCCAAGCTTAGACTACCC-3'; SIG1-RLV,
5'-CGCGGATCCCGTCTTGTTGCGCAGGAAGTT-3'; SIG1-RQR, 5'-CGCGGATCCAGGCAACGTATACATCATGGC-3'; SIG2-STE,
5'-CGCGGATCCTCTACTGAGAAGCCA-3'; SIG2-VRG,
5'-CGCGGATCCGTTAGAGGTTATGTGAAAGGT-3'; SIG3-MLV, 5'-CGCGGATCCATGCTGGTCTTTGTACATCCTC-3'; and SIG3-GLR
(5'-CGCGGATCCGGTTTAAGAACTACATGGAAC-3'. Primers corresponding
to the 3' ends were as follows: SIG1,
5'-CCGAGCTCTTATGATTGTGCAACCAAGTA-3'; SIG2,
5'-CCGGAATTCTCAATTCTTAAGGATCATTGC-3'; and SIG3,
5'-ACGCGTCGACGACATAAGAGTCATTGGAGCC-3'.
PCR products were digested with BamHI, SalI,
SacI, or EcoRI (primer sequences corresponding to
the added restriction sites are in boldface letters) and cloned into
the corresponding sites of the expression plasmid pET28a (Novagen) and
pQE30 (Qiagen) to generate pMA plasmids. Plasmids were amplified in
E. coli BL21 (DE3, pET28a) or M15 (pQE30). After induction
of protein expression with isopropyl
Construction of Hybrid Sigma Factors and Complementation of E. coli rpoD Mutants--
cDNAs corresponding to full-length hybrid
sigma factors were produced by PCR recombination (25) using four
different oligonucleotides for each of the constructs. The 5'
plant-specific primers were as indicated above (SIG1-KLR, SIG2-STE, and
SIG3-MLV), and the 3' E. coli rpoD-specific
primer was 5-GGGGTACCTATGGAGCAAAACCCGCAGTCAC-3', with
an added KpnI site marked in boldface letters. The
complementary recombination primers were as follows:
SIG1/rpoD, 5'-gagatggttgaagcgAACATTCGACTCGTT-3', 5'-AACGAGTCGAATGTTcgcttcaaccatctc-3'; SIG2/rpoD,
5'-gagatggttgaagcgAATGTGCGTTTGGTT-3', 5'-AACCAAACGCACATTcgcttcaaccatctc-3'; SIG3/rpoD,
5'-gagatggttgaagcgACCCGGTCTTTGGTT-3', 5'-AACCAAAGACCGGGTcgcttcaaccatctc-3'. The parts of the primers that
correspond to the principal sigma factor of E. coli are
shown in lowercase letters.
After cleavage with the appropriate restriction enzymes, the PCR
fragments were inserted into Bluescript KS vectors to give pMA plasmids.
For complementation studies E. coli strains UQ285 and CAG1
were obtained from the E. coli Genetic Stock Center (Yale
University, New Haven, CT) and transformed with the corresponding
plasmids. Transformants were selected on L-broth-ampicillin
plates at 32 °C and screened at 42 °C.
In Vitro Transcription--
In vitro transcription
reactions were performed at 37 °C in 25-µl assays. The reaction
mixture contained 40 mM Tris/HCl (pH 8.0); 20 µM dithiothreitol; 14 mM MgCl2;
40 µM EDTA; 50 mM NaCl; 300 µM
each of GTP, ATP, and CTP and 5 µM UTP, including 20 µCi of [ Western Blot Analysis of A. thaliana Proteins--
Antibodies
were produced in rabbits, and horseradish peroxidase-coupled secondary
antibodies were detected using the ECLTM Western blotting
detection system (Amersham Pharmacia Biotech).
Overproduction of Entire and Truncated Plant Sigma Factors in E. coli--
We have cloned and sequenced the cDNAs corresponding to
six putative A. thaliana sigma factors as described under
"Materials and Methods." We have analyzed the function of three of
them (named SIG1, SIG2, and SIG3, according to Ref. 9) in more detail. To this end, selected full-length clones have been used to amplify the
coding regions and to clone them into two different expression vectors,
pET28a and pQE30. Regions coding for supposed transit peptides have
been eliminated in the cloning step, thus locating the N-terminal three
amino acids to the sequences KLR, STE, and MLV, respectively, as
indicated in Fig. 1A.
Following transformation of E. coli BL21 or M15 and
induction with isopropyl-1-thio-
Having thus shown that the N-terminal parts of two plant sigma-like
factors might be incompatible with the E. coli
transcriptional machinery, we recloned only the 3' portions of the
three plant cDNAs that code for three essential functions of a
sigma factor: promoter recognition (regions 2.4 and 4.2), DNA melting
(region 2.3), and RNA polymerase-core interactions (regions 2.1 and
3.2, reviewed in Ref. 8). It has already been shown that truncated sigma factors retain DNA binding and core RNA polymerase binding activity (16-18). Therefore, we assumed that it should be possible to
use such truncated proteins to characterize these two functions by
in vitro transcription assays. Sequence alignment of the
amino acid sequences of the three plant sigma factors with the
one of E. coli shows that the C-terminal regions, but not
the N-terminal parts, of the proteins are highly conserved. Each of
these truncated proteins (detailed in Fig. 1A) could be
produced in large amounts in E. coli, indicating that the
N-terminal parts of SIG1 and SIG3 are responsible for the failure of
overproduction of the entire proteins.
Characterization of Truncated Sigma Factors by in Vitro
Transcription--
SIG2 was the only sigma factor that could be
obtained as an entire protein by overproduction in E. coli.
We used this protein to analyze whether our assumption was correct,
i.e. that the truncated proteins could be used to
characterize the two essential functions of a sigma factor: promoter
recognition and RNA polymerase-core interaction. To this aim we
compared the full-length SIG2 factor (SIG2-STE) with its truncated form
(SIG2-VRG) in an in vitro transcription assay using three
different plant plastid promoters with the E. coli core
enzyme (Fig. 2). The sequences of the
three different plastid promoters and their comparison to the E. coli consensus promoter sequence, which is recognized by the
E. coli primary sigma factor, RpoD, are shown in Fig.
1C. These particular plastid promoters have been chosen for
the following reason: The rbcL promoter represents one
highly transcribed plastid promoter, regulating the transcription of
the most abundant chloroplast protein and thus implying that it should
be recognized by a primary sigma factor. The two other plastid
promoters, rrn-P1 and rrn-P2, have plastid-specific regulatory functions in the expression of the rrn operon (4). Therefore, they represent good candidates to be recognized by an alternative sigma factor that fulfils specific functions in the down-regulation of rrn expression during
plastid development.
The E. coli holoenzyme recognizes all three plastids
promoters in in vitro transcription assays (Fig. 2,
lanes 15 and 18), albeit with different
efficiencies (rbcL>rrn-P2>rrn-P1;
note that rrn transcription is exposed four times longer
than rbcL transcription). The E. coli core enzyme
produces a longer RNA than the one that is initiated at the -180
rbcL promoter (Fig. 2, lanes 1 and 8, asterisk). We did not map the exact location of the 5' end
of this RNA, but the production of this RNA can serve to verify the saturation by sigma factor of commercially available E. coli
RNA polymerase preparations (see Fig. 6, lanes 1-3 and
18). Reconstitution of the E. coli core enzyme
with either the full-length (Fig. 2, lanes 1-7; exposure
times were 12 h for lanes 1-4 and 2 days for lanes 3', 4', and 5-7) or the truncated form of
SIG2 (Fig. 2, lanes 8-14; exposure time was 2 days) gave
similar results. The nonspecific transcription product that was
obtained when using the cloned rbcL promoter as template
diminished gradually with increasing SIG2 concentrations, and very
small amounts of the specific transcript (-180) were produced. The
appearance of the specific transcript does not quantitatively correlate
with the disappearance of the nonspecific transcript. We interpret this result to indicate that the factor has a high affinity for the E. coli core enzyme but a very low affinity for the -180
rbcL promoter. A slightly different result was obtained for
the rrn promoter region. Neither of the two SIG2 proteins
allows initiation, even at low levels, at either of the two E. coli-like rrn promoters (Fig. 2, lanes 5-7
and 12-14). If SIG2-VRG is added to transcription assays
that are performed with the E. coli holoenzyme, the
transcription that initiates at the rrn-P1 promoter is
specifically diminished (Fig. 2, lanes 15-20). This
suggests that of the three promoters tested, SIG2 recognizes only the
rrn-P1 promoter efficiently. The full-length form of SIG2,
SIG-STE, gave the same result as the truncated protein (not shown).
Altogether, this indicates that SIG2 interacts with the E. coli core enzyme. This sigma factor can specifically recognize the
rrn-P1 promoter, but it cannot activate transcription
efficiently, not even when present as a full-length native protein.
In the case of SIG1, we analyzed two different truncated forms of the
protein (RLV and RQR; see Fig. 1). SIG1-RLV corresponds in length to
the truncated form of SIG2 and harbors a supposed 1.2 region at its N
terminus. We found that only SIG1-RLV fulfils a sigma-like function in
the in vitro transcription assay (Fig. 3, lanes 1-16; exposure time
was 12 h). SIG1-RLV does not diminish the production of the
nonspecific RNA (Fig. 3, asterisk) by the E. coli
core enzyme, i.e. SIG1 has much less affinity to the
E. coli core enzyme than SIG2. However, activation of
transcription at the -180 rbcL promoter by SIG1 is much
stronger than that obtained by SIG2 (compare Fig. 2, lanes
1-4, with Fig. 3, lanes 13-16; note that all lanes
were exposed for 12 h). SIG1 recognizes only the -180
rbcL promoter (Fig. 3, lanes 13-16, 12 h of
exposure; lanes 17-19, 4 h of exposure) but does not
recognize either of the two E. coli-like rrn
promoters (Fig. 3, lanes 1-4 and 9-12, 12 h of exposure; lanes 20-22, 4 h of exposure).
Addition of SIG3 (GLR) to the in vitro transcription assays
did not change the transcription pattern obtained with the E. coli core enzyme (not shown), suggesting that the truncated form of the factor does not interact with any of the three tested promoter regions or with the core RNA polymerase.
Analysis of the Promoter Specificity of Plant Plastid Sigma-like
Factors by Complementation of E. coli SIG3 Might Contain an Inhibition Domain--
The low, but obvious,
complementation of the two E. coli rpoD mutants by SIG3 was
surprising when compared with the complete absence of specific
initiation that was observed by in vitro transcription. Therefore, we compared the amino acid sequences of two C-terminal parts
that have been used for either in vivo complementation or for in vitro transcription in more detail. Sequence
alignment with the prokaryotic sigma factors known thus far shows
sequence similarity to the sigma K factor of Bacillus
subtilis in a region of about 100 amino acids (Fig.
5). Sigma K is synthesized as a precursor
protein with a 20-amino acid pro sequence. In the presence of this
sequence, the factor is inactive, and its activation is brought about
by proteolytic removal of the pro sequence (22). The two SIG3
constructs that have been used for in vitro transcription and in vivo complementation differ in the presence and
absence, respectively, of the region that is similar to the inhibiting pro sequence (see Fig. 5). Therefore, low level of complementation could be related to the absence of this domain.
To test this hypothesis, we produced two additional N-terminal
truncated proteins. One of them lacks only the supposed pro peptide
(SIG3-ASL), and the other one lacks the entire N-terminal part that has
amino acid sequence similarity to Sigma K (SIG3-HTR). We performed
reconstitution assays of the E. coli core enzyme with the
two different truncated forms of SIG3 using the same templates as
already described. SIG3-ASL does not affect in vitro transcription (not shown). However, SIG3-HTR stimulates specific initiation at all three promoters, rrn-P1,
rrn-P2, and rbcL (Fig. 6, lanes 4-9; 12 h of
exposure). Transcription of the cloned rDNA promoter produces many
different transcripts. Therefore, we confirmed the P1- and P2-initiated
RNAs by transcribing three different mutated promoter regions. These
mutations have been previously constructed in our laboratory to analyze
the regulation of plastid rDNA transcription by the transcription
factor CDF2 (Ref. 4; Fig. 6, lanes 10-17; 2 days of
exposure). As already shown by transcription of these three templates
using E. coli holoenzyme (4), the mutation in the -35
region of the P2 promoter diminishes initiation at P2 (Fig. 6, compare
lanes 11 and 13). The mutation in the -10 region
abolishes initiation at P2 (Fig. 6, compare lanes 11 and
15), and the mutation in the CDF2 binding site abolishes initiation at the P1 promoter (Fig. 6, compare lanes 11 and
17). Competition assays of SIG3-HTR with the E. coli holoenzyme confirm the recognition of all three promoters by
SIG3-HTR (Fig. 6, lanes 18-23; 2 h of exposure).
Activation of SIG3 Is Not Regulated by Proteolytic Cleavage of the
Hypothetic Inhibition Domain--
To analyze whether the activity of
SIG3 might be regulated in vivo by proteolytic cleavage of
the N-terminal region, we have prepared antibodies against the SIG3-HTR
protein. These antibodies specifically recognize SIG3. They do
not cross-react with SIG1 or with SIG2 (not shown). Western blot
analysis of proteins isolated from Arabidopsis cotyledons
revealed only one polypeptide of about 60 kDa, corresponding to the
size of the full-length protein (Fig. 7).
Three different cDNAs coding for potential plastid-localized
sigma70-type transcription initiation factors have recently
been cloned and sequenced from A. thaliana (9, 23), and the
existence of a multigene family of sigma factors has been suggested for Zea mays (24). Meanwhile, the list of Arabidopsis
sigma-like factors has been extended to six (SIG1- SIG6; see accession
numbers listed above). Considering the small size of the plastid genome compared with that of bacteria or cyanobacteria, the existence of
several sigma-like factors in plastids is quite surprising. It suggests
that these factors might play different roles in plastid gene expression.
The sigma70 transcription factor family is well
characterized in prokaryotes such as E. coli and B. subtilis. It is subdivided into two (8) or three (25) groups
including primary and alternative sigma factors. In the present study,
we analyzed whether the two-group system has been conserved in higher
plant plastids, i.e. whether a functional specialization
might explain the existence of several sigma-like factors in
photosynthetically active plastids. We have used in vitro
and in vivo approaches to study the evolutionary conservation of components of the transcriptional machinery between prokaryotes and higher plant plastids and to characterize the function(s) of three of the six different plant plastid sigma-like factors. Expression and complementation studies (Figs. 1 and 4) of
full-length and truncated plant plastid sigma polypeptides in E. coli indicate that the functions of the N-terminal parts of the
plant sigma factors differ considerably from those of the corresponding
portion of the principal sigma70 factor of E. coli. High level production of the full-length protein in E. coli was only achieved for SIG2. However, SIG2 does not activate
transcription in a heterologous system with E. coli core enzyme (Fig. 2). This might explain why it was possible to produce this
protein in high amounts in E. coli.
To reveal and analyze specificity in promoter recognition of the three
plant sigma-like factors, we used truncated polypeptides that harbor
only the conserved regions 2-4. In this way, we avoid differences in
promoter-RNA polymerase interactions that may arise from different
functions of the different N-terminal sequences of these factors. In
addition, it has been shown previously that the intact
sigma70 factor of E. coli does not bind to DNA
but that the cleavage of the N-terminal part of the factor transforms
it into a DNA-binding protein (16). Therefore, we supposed that it
should be possible to reveal promoter specificity by using truncated
sigma factors. We analyzed transcription factor-RNA polymerase-promoter
interactions either by reconstitution of the truncated polypeptides
with the E. coli core enzyme or by competition of the
truncated polypeptides with the E. coli holoenzyme followed
by in vitro transcription. These experiments clearly
demonstrate differences in the recognition of three selected plastid
promoters, rrn-P1, rrn-P2, and rbcL. SIG3 recognizes all three promoters, i.e. it is the least
specific of the three regulatory proteins. SIG2 recognizes specifically the rrn-P1 promoter, and SIG1 recognizes only the
rbcL promoter (Figs. 2, 3, and 6). The analysis of several
truncated SIG3 proteins shows that SIG3 might contain an inhibition
domain that is similar to the pro sequence of B. subtilis
sigma K. However, we could not detect proteolytic cleavage of SIG3 in
Arabidopsis. Nevertheless, our experiments suggest a
function of this domain in the regulation of SIG3 activity. Instead of
proteolytic cleavage, the activity of SIG3 might be regulated by
posttranslational conformational changes that modify the accessibility
of this putative inhibition domain.
If we compare the three plastid promoter structures with the consensus
sequence recognized by the principal sigma70 factor of
E. coli, we find that the rbcL promoter has the
highest similarity with the E. coli consensus (Fig.
1C). SIG1 recognizes specifically only the rbcL
promoter, as analyzed by in vitro transcription, suggesting
that SIG1 is the plant analogue to the primary sigma factor of E. coli. This hypothesis is further supported by the in
vivo analysis of the hybrid sigma factors (Fig. 4). For this analysis, the N-terminal part of the E. coli primary
Our results indicate that the three higher plant plastid
sigma70-like proteins have at least three characteristics
in common with their prokaryotic ancestors: 1) they are composed of
specific functional domains; 2) they distinguish between different
promoter structures; and 3) only one of them (SIG1) is capable of
recognizing all essential E. coli promoters that are
recognized by the E. coli principal sigma70
factor in vivo in E. coli. Therefore, we consider
SIG1 to be the plant analogue to the primary sigma factor of E. coli. Interestingly, sequence alignment of all six
Arabidopsis sigma factors to all five sigma factors
localized on the Synechocystis genome (13) shows that SIG1
has the strongest sequence similarity and/or identity to all of the
cyanobacterial sigma factors (Table I).
Thus, SIG1 is the most prokaryotic-like plant sigma factor. The other
plant sigma factors might have evolved in coordination with the
transformation of a unicellular organism, the cyanobacterial ancestor,
into an integrated part of a multicellular organism, the present-day
plastid. The in vivo function of the SIG2-specific
rrn-P1 promoter is still unclear
(4),2 and the activity of
SIG3 might be regulated by posttranslational modification(s).
Therefore, our results suggest specific functions for SIG2 and SIG3
that are related either to plant development and/or changes of
environmental conditions. Experiments are in progress in our laboratory
to analyze the function of the three plant sigma factors during
A. thaliana development using an antisense approach.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside, the His fusion proteins were
recovered as inclusion bodies. After solubilization in lysis buffer (20 mM Tris/HCl, pH 8.0, 100 mM NaCl, 8 M urea) containing 1 mg/ml lysozyme, the fusion proteins
were purified on Ni2+-NTA columns according to the
supplier's protocol (Qiagen). After elution with 20 mM
Tris/HCl, pH 8.0, 100 mM NaCl, 8 M urea and 300 mM imidazole, the proteins were renatured by dialysis
against 50 mM Tris/HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 20% glycerol and stored at 
20 °C
until use. The purity of each protein preparation was checked by
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining of the protein.
-32P]UTP; 71 nM template DNA;
and 0.05 units of E. coli holoenzyme (Roche Molecular
Biochemicals) or 26 nM of E. coli core enzyme (Epicentre Technologies). Sigma proteins were added to give final concentrations of 100, 200, or 300 nM. Reaction mixtures
were preincubated for 5 min without template at 37 °C. Transcription was started by addition of DNA template, and the reactions were stopped
after 15 min by extraction with phenol/chloroform and precipitated with
ethanol. Transcription products were analyzed on 8% acrylamide/urea
gels. The template DNA was used in supercoiled form. It corresponded to
the plasmid pTZ19 harboring the corresponding promoter regions and the
E. coli threonine attenuator (2, 4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, only one of the three transcription factors, SIG2, was expressed in
sufficient amounts to allow purification and subsequent testing by
in vitro transcription assays. SIG1 is expressed only at a very low level (not shown), and the expression of SIG3 seems to be
toxic for the bacteria. This can be concluded from the analysis of
growth curves of transformed bacteria (Fig. 1B). After
induction with isopropyl-1-thio--D-galactopyranoside,
the expression of SIG3 causes growth arrest of E. coli.
Similar results were obtained using the pET28a constructions (not
shown). In order to know which part of SIG3 is toxic for the bacteria,
we subcloned the 5' and the 3' portions of the cDNAs. These two
constructions give rise to truncated proteins corresponding to the
N-terminal part (between MLV and GLR) and the C-terminal part (GLR up
to the C terminus) of the factor. We found that the N-terminal part was
sufficient to provoke growth arrest of E. coli (not shown).
The truncated protein corresponding to the C-terminal part could be
produced in large amounts without any harmful effects for E. coli.
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Fig. 1.
Comparison of components of the plant plastid
"E. coli-like" and the E. coli
transcriptional machineries. A, schematic
representation of the principal sigma70 factor of E. coli and of the three putative A. thaliana plastid
sigma-like factors, SIG1, SIG2 and SIG3. The localization of the
conserved domains is indicated by filled, shaded, or
patterned boxes as shown, and the N-terminal ends
of the full-length or truncated plant proteins, which are used in the
following studies, are marked by vertical arrows shown with
the three N-terminal amino acids. B, growth curves of
E. coli cultures transformed with expression vectors coding
for plant sigma-like factors. The time of induction by
isopropyl-1-thio- -D-galactopyranoside is indicated by a
vertical arrow. C, sequence comparison of the
three plastid E. coli-like promoter structures that are used
in these studies with the consensus sequence of the principal E. coli sigma70 factor.
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Fig. 2.
Analysis of promoter-SIG2-RNA polymerase
interactions by in vitro transcription. Plasmids
harboring the cloned rbcL (lanes 1-4, 8-11, and
15-17) or rrn (lanes 5-7, 12-14,
and 18-20) promoter regions were transcribed by E. coli core enzyme (lanes 1-14) or E. coli
holoenzyme (lanes 15-20) supplemented with increasing
amounts of full-length (lanes 2-4, 3', 4', 6, 7 or
truncated (lanes 9-11, 13, 14, 16, 17, 19, and
20) SIG2 protein. The positions of the -180 rbcL
promoter and of the rrn P1 and P2 promoters are indicated by
arrows at the left and right. The
asterisk indicates the position of a nonspecific transcript
produced by the E. coli core enzyme from the rbcL
plasmid. Exposure times were 1 h (lanes 15-17), 4 h (lanes 18-20), 12 h (lanes 1-4), and 2 days (lanes 3', 4', and 5-14).
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Fig. 3.
Analysis of promoter-SIG1-RNA polymerase
interactions by in vitro transcription. Plasmids
harboring the cloned rrn (lanes 1-4, 9-12, and
20-22) or rbcL (lanes 5-8, 13-16,
and 17-19) promoter regions have been transcribed by
E. coli core enzyme (lanes 1-16) or E. coli holoenzyme (lanes 17-22). The transcription
reactions were supplemented with increasing amounts of SIG1-RQR
(lanes 2-4 and 6-8) or SIG1-RLV (lanes
10-12, 14-16, 18, 19, 21, and 22). The positions of
the -180 rbcL promoter and of the rrn P1 and P2
promoters are indicated by arrows at the left and
right. The asterisk indicates the position of an
nonspecific transcript produced by the E. coli core enzyme
from the rbcL plasmid. Exposure times were 12 h
(lanes 1-16) and 4 h (lanes 17-22).
70
Mutants--
To study in vivo the function of the three
plant sigma-like factors, we used two different E. coli
strains harboring thermosensitive rpoD mutations, UQ285 (19)
and CAG1 (20). In both cases, we obtained identical results. Fig.
4 shows the results obtained with the
strain CAG1. To make the plant factors functionally comparable for its
usage in the E. coli system, we fused the 3' ends of the plant cDNAs, coding for the regions 2-1 up to 4-2, to the 5' end of
the primary E. coli 70 factor (Fig.
4A). These constructs result in the production of three
hybrid sigma factors that are identical to the primary
70 factor of E. coli in their N-terminal part
(region 1) but different in the C-terminal parts (regions 2-4). We
found that of the three different hybrid factors (Fig. 4A),
only one (pMA 55, N'-E. coli 70-C'-plant
SIG1; Fig. 4B) complements the E. coli rpoD
mutant strain with the same efficiency as does the homologous E. coli 70 factor (pMRG1, Ref. 21), i.e.
SIG1 represents a primary sigma factor when assayed in E. coli. As a control, we also attempted to complement the two
E. coli mutations with the three full-length plant sigma
factors. All of these assays were negative, indicating that the
plant-specific N-terminal part of SIG1 prevents the complementation. In
Fig. 4B, only the result obtained with full-length SIG1 is shown (pMA 40).
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Fig. 4.
Complementation of E. coli
rpoD mutants with hybrid sigma factors. A,
schematic representation of the three hybrid sigma factors being
composed in the N-terminal half of the E. coli principal
sigma70 factor and in the C-terminal half of the three
A. thaliana sigma factors. The N-terminal borders of the
A. thaliana sigma factors are indicated by a vertical
arrow and the three most N-terminal amino acids (NIR, NVR, and
TRS). pMA 55, 58, and 59 are the plasmids used
for complementation. B, complementation of E. coli strain CAG1 harboring the rpoD800(ts)
mutation with the plasmids pMA 55, 58, and 55. The plasmid pMRG1
corresponds to the control plasmid harboring the cDNA coding for
the E. coli principal 70 factor, and the
plasmid pMA 40 corresponds to the SIG1-KLR control.
View larger version (25K):
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Fig. 5.
Sequence alignment of SIG3 and sigma K. The pro sequence of sigma K is underlined, and the cleavage
site is indicated by a vertical arrow. The 5' ends of the
truncated SIG3 proteins that have been used for in vitro
transcription and in vivo complementation assays are labeled
by stars. The 5' ends of the two supplementary SIG3
truncated proteins that have been used to analyze the function of the
hypothetical inhibition domain are boxed.
View larger version (53K):
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Fig. 6.
Analysis of promoter-SIG3-RNA polymerase
interactions by in vitro transcription. Plasmids
harboring the cloned rbcL (lanes 1-6 and
18-20) or rrn (lanes 7-17 and
21-23) promoter regions were transcribed by the E. coli core enzyme (lanes 2-17) or holoenzyme
(lanes 1, 2, and 18-23) supplemented with the
truncated SIG-HTR protein (lanes 5, 6, 8, 9, 11, 13, 15, 17, 19, 20, 22, and 23). The positions of the -180
rbcL and of the rrn P1 and P2 promoters are
indicated by arrows at the left and
right, respectively. The asterisk shows the
position of the nonspecific transcript that is produced by the E. coli core enzyme from the rbcL plasmid. -35,
-10, and CDF2 correspond to the mutations 2, 3, and 1, respectively, as published by Iratni et al. (4).
WT, wild type. Exposure times were 4 h (lanes
1-3), 12 h (lanes 4-9), 2 days (lanes
10-17), and 2 h (lanes 18-23).
View larger version (28K):
[in a new window]
Fig. 7.
Western blot analysis of A. thaliana cotyledon proteins. 40 µg of total proteins
were separated by SDS-polyacrylamide gel electrophoresis, transferred
to a nitrocellulose membrane, and analyzed by antibodies that have been
prepared against SIG-HTR (left lane) or preimmune serum
(right lane).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 factor (region 1) was fused to the different
C-terminal parts of the three plant sigma factors (regions 2-4).
Region 1 had been shown to be important for open and ternary complex
formation (26) and to induce conformational changes into the holoenzyme
that are important for correct promoter recognition (27). Parts of regions 2 and 4 are important for the recognition of consensus -10 and
-35 DNA sequences (for review, see Ref. 8). Thus, differences in
promoter recognition and initiation of these hybrid sigma factors ought
to be due to differences in the 3' plant-specific part of the
constructs. Results show that of the three tested hybrid sigma factors,
only SIG1 fully complements the E. coli thermosensitive rpoD mutants.
Amino acid sequence similarities and identities between cyanobacterial
and plant factors
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to H. Geiselmann for helpful discussions during the preparation of the manuscript, and we thank H. Pesey for photographic work.
| |
FOOTNOTES |
|---|
* This work was supported by the Region Rhone-Alpes in the frame of the programs Emergence and Biotechnologie and by the European Community in the frame of the Biotech Program BIO4-CT97-2245.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
33-04-76-63-57-44; Fax: 33-04-76-51-43-36; E-mail:
Silva.Lerbs-Mache@ujf-grenoble.fr.
2 S. Lerbs-Mache, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviation used is: PCR, polymerase chain reaction.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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