Báo cáo khoa học: DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression doc

DNA-binding and transcription characteristics of three cloned sigma
factors from mustard (
Sinapis alba
L.) suggest overlapping
and distinct roles in plastid gene expression
Anke Homann and Gerhard Link
Plant Cell Physiology and Molecular Biology, University of Bochum, Germany
We have isolated and studied the cloned sigma factors
SASIG1-3 from mustard (Sinapis alba). In functional ana-
lyses using both promoter and factor mutants, the three
recombinant proteins all had similar basic properties but
also revealed differences in promoter preference and
requirements for single nucleotide positions. Directed muta-
genesis of SASIG1 identified critical residues within the
conserved regions 2.4 and 4.2 necessary for binding of the
)10 and )35 promoter elements, respectively. SASIG1 and
2, but not SASIG3, each have a typical region 2.5 for binding
of the extended )10 promoter element. SASIG3 has a pro-
sequence reminiscent of r
K
from Bacillus subtilis, suggesting
that proteolytic cleavage from an inactive precursor is
involved in the regulation of plastid transcription. In addi-
tion, SASIG2 was found to be more abundant in light-
grown as compared to dark-grown mustard seedlings, while
the converse was true for SASIG3.
Keywords: plastid; promoter; RNA polymerase; sigma fac-
tor; transcription.
Chloroplasts contain the photosynthetic machinery, which
is built-up and maintained by gene-regulatory mechanisms
both inside and outside the organelle. At the level of

transcription this involves the participation of multiple
RNA polymerases, at least two of which are located within
the plastid compartment: (a) a single-subunit type enzyme
related to those of T-odd phages and mitochondria; and
(b) a multisubunit form resembling those of bacteria and
eukaryotic nuclei (reviewed in [1]). The former is a product
of nuclear gene(s) (nuclear-encoded phage-type plastid
RNA polymerase; NEP); the latter, which is the primary
enzyme for transcription of photosynthesis-related chloro-
plast genes [2], contains an organelle-encoded core of
eubacterial a, b, and b¢ homologues [3] and hence has been
termed PEP [4].
The catalytic core of the PEP enzyme assembles with
regulatory proteins that have been identified as functional
equivalents of bacterial sigma factors and hence named
SLFs (sigma-like factors) (reviewed in [5]). Given the central
role of sigma in prokaryotic transcription initiation [6,7], and
considering the endosymbiotic origin of chloroplasts [8], one
might expect coding information for sigma-like protein(s) on
plastid DNA. However, extensive sequence analysis has
shown this not to be the case [3], suggesting that the SLFs
might be nuclear gene products. Direct evidence for this was
obtained by the cloning of sigma-like cDNA sequences from
algae [9,10] followed by those from several higher plants
(reviewedin[11]).
Our previous work on chloroplast transcription in
mustard (Sinapis alba) had resulted in the purification and
biochemical characterization of three SLFs [12,13]. As part
of our efforts to clarify the role of this transcription factor
family, we reported on the cloning of a first member, which

we referred to as SASIG1 [14]. Here we present SASIG2
and SASIG3, and we describe functional studies with all
three recombinant factors.
Materials and methods
PCR and cDNA cloning
Oligonucleotides were derived from expressed sequence
tags with sequence similarity to the coding regions of
the AtSig2andAtSig3 genes from Arabidopsis thaliana
(GenBank accession numbers AC003981 and N97044)
Two pairs, 5¢-GAGAAACAAGTGATACGTTGGA
GA-3¢ and 5¢-TTTCCTGAATGCAGATGACTCTAT-3¢
from AtSig2 and 5¢-GTAGAGATGAACTGGTGAA
AAGCA-3¢ and 5¢-AAAACTTGTAACCCCTTGTGT
GAT-3¢ from AtSig3, were used for PCR-amplification
from total S. alba DNA. The products were cloned into
the EcoRV site of pBluescript (Stratagene), resulting in
plasmids pBS/3C1Sig2 and pBS/4B2Sig3. The inserts were
used for screening of a HybriZAP cDNA library [14].
Clones pAD/3C1Sig2 and pAD/4B2Sig3 contained the
full-length SaSig2 and SaSig3 cDNAs, respectively.
Correspondence to G. Link, Plant Cell Physiology and Molecular
Biology, University of Bochum, Universitaetsstr. 150,
D-44780 Bochum, Germany.
Fax: +49 234 3214 188, Tel.: +49 234 322 5495,
E-mail:
Abbreviations: EMSA, electrophoretic mobility shift assay; HTH,
helix-turn-helix; NEP, nuclear-encoded phage-type plastid RNA
polymerase; PEP, bacterial-type plastid RNA polymerase with core
subunits encoded by organellar genes; SLFs, sigma-like factors.
Enzyme: DNA-dependent RNA polymerase (EC 2.7.7.6).

(Received 19 November 2002, revised 23 January 2003,
accepted 3 February 2003)
Eur. J. Biochem. 270, 1288–1300 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03494.x
Isolation of proteins and Western blot analysis
Mustard (S. alba) seedlings were grown at 25 °Cfor4days
either under continuous light (250 lmol photonsÆm
)2
Æs
)1
)or
in the dark. Cotyledons were harvested and whole-cell or
plastid proteins were prepared as described [15,16]. Bacteri-
ally expressed recombinant proteins were isolated from
inclusion bodies, followed by SDS/PAGE. The gel-purified
proteins were used as antigens in a custom protocol for rabbit
immunization (Eurogentec). For Western analysis, protein
samples were separated by SDS/PAGE and transferred
onto nitrocellulose membrane. Proteins were incubated
with anti-SASIG2 and anti-SASIG3 antisera, respectively,
and detected by nitroblue tetrazolium/5-bromo-4-chloro-
3-indolylphosphate.
Cloned fragments of mustard chloroplast DNA [5,14,17]
Plasmid pSA05/H120 carries the psbA promoter (accession
no. X04826); the 120-bp HinfI insert (H120) covers 68 bp
upstream of the transcription start site and 52 bp noncoding
5¢-sequence of the gene. A 450-bp EcoRI–HindIII fragment
that carries the trnK (X04826) promoter was prepared from
pSA364-EH450, a pSPT18-based derivative of pSA364.
Bam0.5, the 460-bp BamHI fragment of pSA364-B0.5,
represents intron sequences of the split trnK gene (X04826).

Plasmid pSA364-ET0.2 contains the trnQ (X13558) pro-
moter and flanking sequences in pSPT19. The 213-bp
fragment resulting from cleavage by EcoRI and TaqIcovers
56 bp downstream of the transcription start site. The
pUC13-based plasmid pSA364-H018 carries the rps16
(X13609) promoter; its 188-bp HinfI insert extends 107 bp
downstream of the transcription start point. The rbcL
plasmid pBS1.4E/P (X73284) was constructed by cloning a
1.4-kb EcoRI–PstI fragment of pSA530 into pBSIISK.
Using BamHI and HindIII digestion, an  500-bp subfrag-
ment containing the rbcL promoter and flanking sequences
(164 bp downstream of the transcription start site) was
generated. Plasmid pBS-1.7kb-B, including the promoter
and both coding and noncoding regions of the ycf3 gene
(AJ242660), was cleaved with DdeIandEcoRV; this
resulted in a 273-bp fragment that covered 158 bp down-
stream of the ycf3 transcription start. The 205-bp insert of
pBSKS/205TD carries the region upstream of the rrn16
gene (X04182) that contains the P1, PC and P2 promoters
[3]. All DNA fragments specified above were used as
unlabelled competitors in electrophoretic mobility shift
DNA binding assays (EMSA). In addition, the H120
fragment that carries the psbA promoter was used as a
labelled probe and for construction of point mutants, and
the EcoRI-linearized pSA05/H120 served as a transcription
template (see below). The DNA fragments that carry
mustard chloroplast promoters are summarized in Table 1
and details of the promoters are depicted in Figure 5A and
6A.
Bacterial expression and purification of recombinant

factors
The SaSig1–3 cDNAs lacking the transit peptide region
were each cloned in pQE30 (Qiagen). A truncated SaSig3
construct was made by using the PCR primers 5¢-CACA
CAAGGGGTTACAAGTTCTCCACG-3¢ and 5¢-ACCA
GCCAATTGGTTCCAAAAATCTATCT-3¢ and ligation
into pQE31 (Qiagen). Following transformation of M15,
the recombinant proteins were purified on Ni-nitrilotriacetic
acid agarose columns (Qiagen).
Gel shift DNA-binding assays
Recombinant sigma factors were incubated with 2.5 ng
32
P-labelled H120 fragment and 0.5 lg E. coli core RNA
polymerase in 50 lLof30m
M
Tris/HCl pH 7.0, 5 m
M
b-mercaptoethanol, 0.5 m
M
EDTA, 5% (v/v) glycerol for
10 min at 25 °C. DNA–protein complexes were analysed on
a native 5% (w/v) polyacrylamide gel (29 : 1 acrylamide/
bisacrylamide) containing 0.5
M
Tris/HCl pH 8.8. The gel
was dried and then analysed using a Fuji BAS 2040-
phosphoimager. Unlabelled DNA fragments representing
various chloroplast promoters were prepared as summar-
ized in Table 1. These fragments were used in competition
EMSA (Fig. 5). Likewise, psbA promoter fragments carry-

ing point mutations were used as competitors (Fig. 6).
Mutagenesis of the psbA promoter
The cloned 120-bp HinfI region containing the psbA
promoter [5] (see Table 1) was used as the starting
material for the construction of promoter point mutants.
M-19 A/G, M-21 A/T, M-22 T/A, M-23 A/T and M-34
T/C were obtained by the M13-based technique previ-
ously described [18]. All other point mutants were made
by PCR using the QuickChange site-directed mutagenesis
kit (Stratagene). Both the length and sequence outside the
changed position were confirmed to be identical for each
mutant fragment.
Mutagenesis and expression of SASIG1-300Q/H
and SASIG1-455R/H
SASIG1 mutants were constructed by using the Quick-
Change mutagenesis kit (Stratagene). Primers for the
substitutions 300Q/H and 455R/H were 5¢-TATACTGG
TGGATTCGACACGGTGTGTCAAGAGCATTAG-3¢
and 5¢-GAGAGAGAGGGTTCATCAGGTGGGGCTT
GTGG-3¢, respectively. The fragments were cloned into
the BamHI/SalI sites of pMAL-c2x (NEB). The bacterially
Table 1. Cloned DNA fragments containing mustard chloroplast pro-
moters. Plasmids were digested using the indicated restriction enzymes.
DNA fragments were gel-purified and then tested in competition
EMSA experiments as described in Materials and methods.
Gene
Fragment
size (bp)
Restriction
enzyme(s) used

Portion downstream
of transcription start (bp)
psbA 120 HinfI 52
trnK 450 EcoRI/HindIII 352
trnQ 213 EcoRI/TaqI56
rps16 188 HinfI 107
rbcL 500 BamHI/HindIII 164
ycf3 273 DdeI/EcoRV 158
rrn16 205 DdeI/TaqI 140
Ó FEBS 2003 Recombinant plastid sigma factors (Eur. J. Biochem. 270) 1289
expressed mutant factors fused to maltose binding protein
were purified on amylose affinity columns (NEB).
In vitro
transcription
In vitro transcription reactions (25 lL) contained 50 m
M
Tris/HCl pH 8.0, 80 m
M
(NH
4
)
2
SO
4
, 10 m
M
MgCl
2
,1m
M

dithiothreitol, 600 l
M
each of ATP, GTP and CTP, 10 l
M
UTP, 20 lCi [a-
32
P]UTP (Amersham, 400 lCi mmol
)1
),
10 U RNaseOUT (BRL), 0.05 U of E. coli RNA poly-
merase holoenzyme (Roche) or 25 n
M
core enzyme (Epi-
centre), and 1 lg double-stranded EcoRI-linearized DNA
(pSA05/H120 carrying the psbA promoter; see Table 1 and
section ÔCloned FragmentsÕ above). Sigma proteins were
added to give a final concentration of 100 n
M
. Following
preincubation at 30 °C for 10 min without template, the
latter was added and incubation was continued for 15 min.
After phenol/chloroform extraction and ethanol precipita-
tion the transcripts were electrophoresed on 6% (w/v)
sequencing gels.
Results
Characterization of cDNAs for putative sigma factors
from
S. alba
The full-length cDNAs for SASIG1 [14] (accession number
Y15899), SASIG2 (this work; accession number AJ276656)

and SASIG3 (accession number AJ276657) were cloned
from a mustard cDNA library. They translate into open
reading frames for 481 (SASIG1), 575 (SASIG2) and 567
amino acids (SASIG3), respectively. As shown in Fig. 1, the
C-terminal portion of each derived SASIG polypeptide
resembles that of eubacterial r
70
-type factors with their
typical regions 1.2–4.2 [19]. Within these regions, sequence
elements can be located for which distinct functions have
been assigned in bacterial systems. This is exemplified in
Fig. 1 for region 4.2, which contains a helix-turn-helix
(HTH) unit [20] involved in recognition of the )35 promoter
element [7].
Compared to the C-terminal portion, the N-terminal half
of the SASIG proteins is less conserved (Fig. 3A). Although
a putative region 1.2 could be localized as depicted in
Fig. 1A and 3A, no definite assignment of a region 1.1 was
Fig. 1. Sequence features of derived putative sigma factors from mustard
(Sinapis a lba L). (A) Upper: Principal regions of eubacterial r
70
-type
factors (left, N terminus; right, C terminus). Lower: Schematic repre-
sentation of SASIG1–3. Boxes show the location of the conserved
regions shared with the eubacterial r
70
family as well as the putative
transit peptide (TP). Amino acid positions are indicated below each
factor and positions of restriction sites within the corresponding
cDNA sequences are shown above. A truncated SASIG3 protein

(SASIG3-374) is indicated below the full-length protein. (B) Sequences
of the putative N-terminal transit peptides of SASIG1, 2 and 3. Serine
and threonine residues are marked by dots, basic residues by + signs.
The hypothetical cleavage sites are marked by arrows. (C) Alignment
of regions 2.1–4.2 of SASIG1 (Y15899, Kestermann et al. 1998),
SASIG2 (AJ276656) and SASIG3 (AJ276657) from S. alba and r
70
from E. coli (U23083). The HTH motif in region 4.2 is boxed.
1290 A. Homann and G. Link (Eur. J. Biochem. 270) Ó FEBS 2003
possible (sequence data not shown). The most proximal
part of each N-terminal SASIG protein revealed character-
istics that could be implicated with chloroplast targeting on
the basis of
CHLOROP
[21],
PSORT
[22],
TARGETP
[23] and
PCLR
[24]. The putative transit peptide was predicted to
comprise 83 amino acids in SASIG1, 39 in SASIG2, and 74
in SASIG3 (Fig. 1B).
Plastid localization of SASIG proteins
Evidence for chloroplast targeting of SASIG1 was previ-
ously obtained [14]. To find out if SASIG2 and 3 are
likewise chloroplast localized, antibodies were raised against
the recombinant proteins and used in immunoblotting
experiments. In brief, cotyledons of light-grown mustard
seedlings were harvested and protein extracts were prepared

from either whole cells or purified chloroplasts. The extracts
were then electrophoretically separated, immunoblotted,
and probed with anti-SASIG2 and anti-SASIG3 sera. As
shown in Fig. 2A, each antiserum detected one single band,
at 61 kDa (SASIG2) and 65 kDa (SASIG3), respectively,
both in the whole-cell (lanes 3 and 6) and chloroplast
fractions (lanes 4 and 7). It was therefore concluded that
both SASIG2 and SASIG3 are to a large part localized to
the chloroplast.
To investigate if the expression of SASIG2 and SASIG3
was affected by light vs. dark growth conditions, mustard
seedlings were grown for 4 days either under continuous
light or in darkness. Whole-cell protein extracts were
prepared from cotyledons and subjected to immunoblot
analysis with antisera raised against SASIG2 or 3. As shown
in Fig. 2B, the authentic plant protein corresponding to
SASIG2 was found in higher relative amounts in the light
(L) then in the dark (D). The converse was true for SASIG3,
which accumulated to higher levels in etioplast-containing
dark-grown tissue, indicating differential expression of the
respective genes under these two growth conditions.
Furthermore, additional distinct bands below the major
signal were visible in the protein sample from dark-grown
tissue (Fig. 2B, lane 4) following incubation with anti-
SASIG3. Although cross-reaction of the antiserum with
other (dark-specific) proteins cannot be ruled out, it appears
more likely that these smaller extra bands resulted from
proteolytic cleavage (see Discussion).
Sigma-like enzymatic properties of the cloned SASIG
proteins

To help decide whether the cloned SASIG proteins had
indeed properties consistent with a role as a sigma factor, we
used gel shift DNA binding (Fig. 3B) and in vitro transcrip-
tion assays (Fig. 4). In each case, the full reaction contained
either of the recombinant proteins mixed with E. coli core
RNA polymerase and the mustard psbA promoter [5,18].
The choice of the heterologous system was based on previous
findings demonstrating that the (biochemically purified)
chloroplast SLFs in combination with E. coli core enzyme
Fig. 2. Chloroplast localization and expression characteristics of SASIG
proteins. (A) Immunodetection of SASIG2 and SASIG3 in protein
fractions from mustard cotyledons. Twenty micrograms whole-cell
proteins (c) and proteins extracted from isolated chloroplasts (cp) were
each immunoblotted using antisera against SASIG2 (lanes 3 and 4) and
SASIG3 (lanes 6 and 7), or the corresponding preimmune sera (pi)
(lanes 2 and 5), respectively. Lane 1 shows the Coomassie-stained
chloroplast protein extract. (B) Proteins extracted from light-grown (L)
or dark-grown (D) 4-day-old seedlings were immunoblotted with either
anti-SASIG2 (lanes 1 and 2) or anti-SASIG3 antisera (lanes 3 and 4).
Ó FEBS 2003 Recombinant plastid sigma factors (Eur. J. Biochem. 270) 1291
were capable of faithful and efficient binding and transcrip-
tion initiation at this promoter [25].
Fig. 3A gives a schematic view of the SASIG1–3 sequences
derived from the full-length cDNAs. The recombinant
proteins used in the in vitro experiments lacked the N-ter-
minal putative transit peptide. Also included in this analysis
was a more extensively truncated derivative of SASIG3,
which represented only the C-terminal portion with position
374 as the first residue (SIG3-374) (Fig. 3A, bottom).
As shown in Fig. 3B, the full reactions containing DNA,

core enzyme and one of the recombinant SASIG proteins
always resulted in a band shift (ÔbÕ position) as compared to
the free probe (ÔfÕ position; lanes 8–11). The binding signal
was stable in the presence of excess nonpromoter DNA, i.e.
the Bam0.5 intron fragment of the mustard chloroplast trnK
gene [14] (lanes 12–15). The signal intensity became highly
reduced, however, if unlabelled psbA promoter fragment
was used as a competitor, as is exemplified in lane 16 for
SASIG3 (see Figs 5 and 6 for the other factors). None of the
controls consisting of the DNA probe alone (lane 1), or of
probe plus recombinant proteins (lanes 4–7), gave a labelled
band at the position of the binding signal. Although a band
was visible at this position when the probe fragment was
incubated with E. coli core enzyme alone in the absence of
poly(dIdC) (lane 3), this unspecific binding signal [13]
almost completely disappeared in the presence of the
polynucleotide (lane 2). These data thus provided initial
evidence that the SASIG proteins were able to confer
specific DNA binding on the E. coli core polymerase.
In another set of experiments we tested psbA promoter-
driven transcription by core polymerase in the presence or
absence of the recombinant SASIG proteins. As shown in
Fig. 4, multiple run-off transcripts were detected following
synthesis with core enzyme alone (lane 2) and none were
visible with any of the recombinant factors alone (data not
shown). In contrast, a major (66-nt) transcript of the size
expected for faithful initiation at the psbA promoter [18] was
visible with E. coli RNA polymerase holoenzyme (lane 1). A
single transcript of this size was detected also in the presence
of core enzyme plus SASIG1 (lane 3), SASIG2 (lane 4), or

SASIG3-374 (lane 6), indicating that each of these factors
had mediated correct initiation. This band was not visible,
however, in the presence of core enzyme plus full-length
SASIG3 (lane 5).
Together, the EMSA (Fig. 3B) and transcription data
(Fig. 4) suggest that the recombinant SASIG proteins
confer promoter-specific binding and transcription
initiation on the RNA polymerase, thus providing clues as
to their functional roles as sigma factors [19]. In the case of
SASIG3, however, this seems true only for the truncated
SASIG3-374, whereas the full-size protein directs DNA
binding (Fig. 3B, lane 10) but not transcription initiation
(Fig. 4, lane 5).
Relative affinity of SASIG proteins to chloroplast
promoters
To further compare the promoter binding characteristics of
the recombinant factors, they were tested in combination
with a number of mustard chloroplast promoters as
depicted in Fig. 5A. Competition gel shift experiments were
carried out in the presence of E. coli core enzyme, labelled
psbA promoter fragment, and excess unlabelled fragments.
As shown in Fig. 5B, the latter varied in their efficiency to
act as competitors, both compared to one another and
depending on which sigma factor was present in the reaction
mixture. The largest decrease in the intensity of the
radioactive binding signal was always found when the psbA
promoter fragment itself was used as competitor, which
reflects the strength of this promoter [18] (Fig. 5A).
Fig. 3. Recombinant SASIG proteins exhibit sigma-like characteristics in DNA EMSA. Gel shift assays with recombinant SASIG factors 1–3 and
SASIG3-374. Each factor was incubated with a

32
P-labelled psbA promoter fragment (H120) in the absence (lanes 4–7) or presence (lanes 8–11) of
E. coli core RNA polymerase. Controls include labelled H120 alone (lane 1), core enzyme without sigma factor in the presence or absence of
poly[dIdC] (lanes 2 and 3), and full reactions (labelled DNA plus core plus sigma) incubated with unlabelled excess competitor DNAs (lanes 12–16).
The latter were either the promoter-less fragment Bam0.5 (lanes 12–15) containing a portion of the trnK intron [14] or the H120 fragment itself
(psbA promoter; lane 16). Positions of free labelled H120 (ÔfÕ) and of protein-DNA complexes (ÔbÕ) are given in the right margin.
1292 A. Homann and G. Link (Eur. J. Biochem. 270) Ó FEBS 2003
For the majority of other tested promoters, including
those for trnK, trnQ, rps16 and rrn16, the competition
patterns were similar with either SASIG1 or SASIG3
(including the truncated form SASIG3-374; data not
shown), and they differed from that observed with SASIG2
(Fig. 5B). The only deviations from this general pattern
were observed for the rbcL and ycf3 promoters (Fig. 5A).
The rbcL promoter was completely ineffective in the
presence of SASIG3 but showed slight competitor efficiency
in the case of either SASIG1 or SASIG2. None of the three
recombinant factors seemed to be able to mediate efficient
binding to the ycf3 promoter as revealed by its almost
complete inactivity as a competitor.
Mutational studies reveal common and distinct
DNA-binding properties of SASIG proteins
To analyse the role of single nucleotide positions within the
chloroplast psbA promoter for DNA-binding, competition
gel shift assays were carried out (Fig. 6B). The psbA
promoter mutants had base substitutions in either of the
conserved regions, i.e. the )35-region, the TATA box-like
element, the )10-region, or the extended )10-motif
(Fig. 6A). The sigma factors that were used included the
SASIG proteins as well as r

70
from E. coli.
Of the psbA promoter mutants that were altered in the
)35-region, M-32 G/A (Fig. 6B, upper right panel) almost
completely lacked competitor efficiency in the EMSA,
suggesting that the G/A exchange at this position had
rendered the psbA promoter inactive as a binding target
with any of the four sigma factors. In contrast, the M-34
T/C mutant (upper row, middle) had a noticeable effect as a
competitor, and even more so did M-30 C/A (second row,
left). The latter revealed strength comparable to that of the
wild-type promoter (WT; upper left) if either of the SASIG
proteins was used. In the presence of r
70
, however, this
mutant had less competitor strength than the wild-type
construct, which may indicate minor differences in binding
requirements at this position for the plant vs. bacterial
factors. Despite this, the results obtained with the )35
mutants support the view that bases within this region of the
psbA promoter are common determinants of DNA binding
strength.
In efforts to apply mutational strategies to other regions
of the psbA promoter, we introduced four different single-
base substitutions into the TATA-box like element
(Fig. 6A). When these mutant constructs were tested, a
partial loss of competitor strength became apparent, and
this effect was more pronounced in the presence of SASIG1
andSASIG2comparedtoSASIG3andr
70

.Thiswasmost
evident in the case of M-19 A/G (Fig. 6B, third row, middle
panel) and M-22 T/A (second row, right), whereas in the
case of M-23 A/T (second row, middle) and M-21 A/T
(third row, left) the decrease in competitor efficiency
compared to wild-type (WT; upper left) was only marginal.
These data suggested that the plant factors SASIG1 and
SASIG2 were capable of recognizing bases within the spacer
between the )10- and )35-regions. In contrast, SASIG3 and
r
70
seemed to be less dependent on contact sites within this
region of the psbA promoter.
When base changes within the )10-region (Fig. 6A)
were tested in the EMSA (Fig. 6B, bottom row), they all
caused a reduction in competitor strength compared to
the wild-type psbA promoter, yet to different extents.
Only a moderate decrease in competitor strength com-
pared to wild-type (WT; upper left) was observed for M-5
T/C, a mutant in which the last base of the )10-element
was altered (bottom row, right). More dramatic effects
were noticeable with either the double mutant M-5–6
T/C–C/A (bottom row, middle) or with M-10 T/A
(bottom, left).
In contrast with the )35 mutant M-32 G/A (upper right
panel), none of these )10 mutations seemed to uniformly
affect binding by all four sigma factors. In the case of M-5–6
T/C–C/A, the competitor strength of this mutant was still
significantly stronger in the presence of SASIG1 than with
any of the other factors tested. The )10 T/A mutation

dramatically affected the promoter usage by SASIG1 and
wild-type; SASIG2 (as well as r
70
), but more weakly that by
SASIG3 (Fig. 6B, lower left). The latter factor lacks the
conserved Glu in the )10-recognition region (Fig. 7A),
Fig. 4. Faithful in vitro transcription from the chloroplast psbA pro-
moter functionally assigns the SASIG proteins as sigma factors. Tran-
scripts generated from the linearized plasmid pSA05/H120 by E. coli
RNA polymerase holoenzyme (lane 1), core enzyme alone (lane 2),
core enzyme plus SASIG1–3 or SASIG3-374 (lanes 3–6). Run-off
transcripts with a size expected for correct initiation at the in vivo start
(+1) are marked by the arrow.
Ó FEBS 2003 Recombinant plastid sigma factors (Eur. J. Biochem. 270) 1293
which participates in binding to the first base of the
)10-element in E. coli [26].
The (5¢-TG-3¢) positions )13 and )12 of the mustard
psbA promoter match those of the extended )10-motif of
bacterial promoters, i.e. the known contact site for residues
in the sigma region 2.5 [27]. Conserved amino acids
reminiscent of this region are present in SASIG1 and
SASIG2 but not in SASIG3 (Figs 7A; H and E). It was
therefore of interest to test the possible role of bases within
this region of the chloroplast promoter. In contrast, the G/T
transversion at )12 (Fig. 6A) resulted in almost complete
loss of competitor efficiency in the presence of SASIG1,
SASIG2 or r
70
(Fig. 6B, third row, right panel). With
SASIG3, however, only a partial reduction in competitor

strength compared to wild-type (upper left) was noticeable,
which is consistent with the lack of a conserved region 2.5 in
this factor (Fig. 7A).
Inthecaseofr
70
the HTH-motif [20] in region 4.2 is
involved in binding of the )35 promoter element [7]. As
shown in Fig. 7A, two conserved amino acids, E13 and
R16, can be identified in all three SASIG proteins within the
HTH-region. In r
70
the Arg corresponding to R16 (R588) is
known to interact with the third base of the )35-element of
prokaryotic promoters [28]. Using SASIG1, we therefore
converted R16 into a His and tested the effect of the
resulting sigma mutant SASIG1–455R/H in competition
EMSAs (Fig. 6). The maltose binding portion of the
recombinant proteins did not interfere with core or promo-
ter binding (data not shown).
As is evident from Fig. 7B (right panels; M-32 G/A), the
mutant promoter M-32 G/A revealed almost wild-type
competitor strength in the presence of the sigma mutant
(455R/H), whereas it was largely inactive in the presence of
wild-type SASIG1. None of the other )35-mutant promo-
ters (Fig. 6A) showed any significant response to SASIG1–
455R/H (data not shown). These results established the
importance of residue(s) within the putative HTH-motif of
the plastid sigma factor, which hence may be functionally
relatedtothatofbacterialr
70

.
To study the interaction with the )10-region, we
converted the Glu (Q300) in SASIG1 into a His and
tested the promoter affinity of the resulting mutant factor
SASIG1–300Q/H (Fig. 7B, panels on the left). Whereas
the mutant promoter construct M-10 T/A almost com-
pletely lacked competitor efficiency in the presence of the
Fig. 5. Promoter affinity of SASIG factors in competition EMSA
studies. (A) Sequence architecture of mustard chloroplast promoters
used as competitors: psbA, trnK, trnQ, rps16, rbcL, ycf3, and rrn16 (P1
promoter). The )10, TATA-like and )35 elements are indicated
(shaded) and transcription startpoints marked by arrowheads. (B)
Competition EMSAs. The labelled psbA promoter fragment H120 was
incubated with recombinant sigma factor in the presence of E. coli core
polymerase, poly[dIdC], and unlabelled promoter fragments. The
reaction mixtures were analysed as in Fig. 3B and the binding signals
were quantified by phosphoimaging. The DNA binding activity in the
presence of competitor DNA (light grey, 25 ng; dark grey, 100 ng) is
expressed as a percentage of the signal intensity relative to that in the
absence of competitor (100%). The data represent mean values of
three independent experiments.
1294 A. Homann and G. Link (Eur. J. Biochem. 270) Ó FEBS 2003
unmodified SASIG1 protein (WT), it showed considerable
strength (binding of the labelled psbA probe only 40% of
that in the control) in the presence of the mutant factor
(300Q/H). Hence this sigma mutant counteracted the effect
of M-10 T/A. None of the other )10 promoter mutations
(Fig. 6) were compensated by the Q/H exchange in
SASIG1, nor was the psbA wild-type promoter affected
to any appreciable extent (data not shown).

Fig. 6. DNA sequence determinants for sigma factor-mediated binding to the psbA promoter. (A) Promoter mutants. The sequence of the wild-type
promoter (WT) is given, with the principal elements indicated on an extra line (top). The small horizontal arrow at +1 depicts the transcription start
site. Positions of base substitutions and names of the resulting variant promoters are shown below. (B) Competitor strength of wild-type and mutant
psbA promoters in EMSA. The basic outline of the experiments was as in Fig. 5, using labelled wild-type psbA promoter and either wild-type or
mutant promoters as unlabelled competitors. Each bar represents the DNA binding activity in the presence relative to that in the absence of 25 ng
(light grey) or 100 ng (dark grey) competitor DNA. The data represent mean values of three independent experiments.
Ó FEBS 2003 Recombinant plastid sigma factors (Eur. J. Biochem. 270) 1295
Discussion
In the present work we have studied the three cloned
mustard factors SASIG1-3. As shown in Figs 1 and 2, all
three proteins have extensive sequence similarity to the
conserved regions 1.2–4.2 of bacterial sigma factors (see, e.g.
[7,29]). In contrast, a sequence that would closely match
region 1.1, i.e. a known modulator of DNA binding by
regions 2 and 4 [30], was not detected.
The N-terminal region of each SASIG protein exhibits
stretches rich in Ser and Thr residues (Fig. 1B), which is a
property of many chloroplast transit peptides (for review
see [31]). and this region was tentatively identified as a
transit peptide by several localization prediction pro-
grams. The predominant chloroplast localization of the
authentic mustard proteins corresponding to SASIG2 and
SASIG3 was verified by immunoblotting experiments
(Fig. 2) and for SASIG1 this had previously been
demonstrated by in organello import assays [14]. We wish
to note that it cannot be ruled out that a minor fraction
might be targeted to different intracellular sites, as was
shown recently for other chloroplast transcriptional
proteins [1,32], including a putative sigma factor from
maize [33]. However, a predominant chloroplast localiza-

tion of the mustard SASIG proteins is in agreement with
the conclusions reached for the Arabidopsis putative
sigma proteins [34–36].
Transcript analyses of sigma factor genes from monocot
and dicot plant species showed most of these to be more
actively expressed under light-grown as compared to dark-
grown conditions in plant tissue [37–41], and to be almost
silent in roots [34,36,42]. On the other hand, Western
analyses have provided evidence for differential expression
profiles at the protein level. Our previous results for
SASIG1 [14] and the present data for SASIG2 and SASIG3
together suggest that the three factors do not have uniform
expression profiles. Both SASIG1 and SASIG2 (Fig. 2B,
lanes 1 and 2) accumulate preferentially in green tissue of
light-grown seedlings, whereas SASIG3 is more abundant in
etioplast-containing dark-grown tissue (lanes 3 and 4). This
situation is reminiscent of the protein accumulation profiles
of two putative sigma factors from the monocot Zea mays.
The ZMSIG3 protein, which has 36% sequence similarity to
SASIG3 (data not shown), was mostly found in nongreen
tissue, whereas ZMSIG1 (50% similarity with SASIG2)
accumulated in green leaf tissue [15].
That the recombinant SASIG proteins have enzymatic
characteristics consistent with a role as a sigma factor was
demonstrated both in EMSA DNA binding (Fig. 3B) and
in vitro transcription experiments (Fig. 4). In the gel shift
assays each of the three proteins was found to interact with
E. coli core enzyme, resulting in a functional RNA poly-
merase holoenzyme that was capable of binding to the
chloroplast psbA promoter (Fig. 3B, lanes 8–11). The

specificity of DNA binding was indicated by the result that
excess unlabelled psbA promoter fragment could function as
a competitor (Figs 5 and 6), whereas complex formation
was resistant to the addition of a nonpromoter fragment
(Fig. 3, lanes 12–15). As shown in Fig. 3, there was no
retarded signal with any of the sigma factors alone in the
absence of core enzyme (lanes 4–7). This was true also for
the N-terminally truncated factor SASIG3-374, in contrast
Fig. 7. Amino acid residues of SASIG factors involved in promotor
binding. (A) Sequence alignments. The regions of r
70
known to be
contact sites for the )35-element (HTH-motif), the )10-element ()10-
recognition region), and the extended )10-element (region 2.5) were
aligned with the SASIG factors and conserved positions are shown
boxed and light grey. Arrows point to the cognate promoter elements
()35/)10; EX, extended )10), here exemplified by those of the psbA
promoter (central line). (B) Results of competition EMSA, showing
activity of promoter mutants M)10 T/A and M-32G/A with either
SASIG1 or the mutant factors SASIG1–300Q/H or SASIG1–455R/H.
The positions of the changed amino acids of SASIG1 are given on an
extra line below.
1296 A. Homann and G. Link (Eur. J. Biochem. 270) Ó FEBS 2003
to the situation for r
70
, which upon cleavage of its N
terminus becomes able to bind to promoter DNA in the
absence of core enzyme [30]. Furthermore, in native r
70
regions 1.1 and 4 are located in close proximity, with region

1.1 acting as an autoinhibition domain. Sigma–core inter-
action induces a conformational change that unmasks the
DNA binding domains [43,44]. The lack of DNA binding
by SASIG3-374 is consistent with the apparent absence of a
functional region 1.1 in (full-length) SASIG3, although it
should be noted that also region 1.2 and part of region 2
were removed during construction of SASIG3-374.
To further clarify the role of the SASIG proteins as
initiation factors, in vitro transcription assays were carried
out. The results depicted in Fig. 4 established this role for
SASIG1 and SASIG2, but not for SASIG3. Transcripts of
the size expected for correct initiation could only be detected
with truncated SASIG3-374 but not with the full-length
protein, indicating that the latter might be inactive because
of inhibitory sequences at the N terminus.
Inspection of the SASIG3 sequence revealed a motif
(residues 277–298) with strong similarity to the amino
terminus of r
K
from B. subtilis [45] (Fig. 8C). It has long
been known that certain sporulation factors (r
E
, r
K
)are
synthesized as inactive precursors that are converted into
the active mature proteins by site-specific proteolysis of
approximately 20 amino acids at the N terminus [46,47]. A
transcription-inhibitory effect of the N-terminal region of
the SASIG3 homologue from Arabidopsis thaliana,

ATSIG3, has recently been described [48]. Our present data
confirm and extend these findings, suggesting that the
SASIG3 N-terminal region inhibits transcription (Fig. 4)
but not promoter binding (Fig. 3B).
If full-length SASIG3 represents the transcriptionally
inactive ÔproÕ form of this plastid sigma factor, the cleavage
and subsequent release of proteolytic fargment(s) might be a
rapid and tightly regulated process involving one of the
known chloroplast proteases in vivo [49]. Our immunoblot-
ting experiments (Fig. 2B, lane 4) suggest limited proteolytic
cleavage of the authentic protein detected by antibodies
against SASIG3. It is interesting to note, however, that
smaller-sized bands were detected only with blotted proteins
from dark-grown seedlings (lane 4), where SASIG3 seems to
be present in higher relative amounts than in the fraction
from light-grown material (lane 3). It is possible that the
kinetics of the proteolytic cleavage differ in a plastid-type
and/or developmental stage-specific way, with immediate
consequences for the availability and function of SASIG3
in vivo as depicted in the model shown in Fig. 8.
This model is based on our observations that full-length
SASIG3 efficiently binds to the promoter DNA but is
unable to initiate transcription. Only after cleavage of the
N-terminal region does the truncated factor (such as
SASIG3-374) become transcriptionally competent. The
enzymatic conversion of the pro-factor into a fully func-
tional (truncated) SASIG3 protein not only has an effect on
transcription under the control of this factor itself, but also
on that driven by other plastid sigma factors. Based on the
similarities in promoter usage in vitro by SASIG1–3 (Figs 3,

5 and 6), it is conceivable that these factors are capable of
competing for one and the same promoter. Tight binding of
one factor (e.g. the SASIG3 pro-factor) hence can result in
inhibition of transcription by others.
Work on the in vivo expression of the sigma factors from
other plants has established overlapping transcript patterns
for individual members [34,37,40]. Moreover, genetic evi-
dence is available from Arabidopsis, where a ATSIG2
knockout mutant has only a weak and stage-specific
chlorophyll-deficient phenotype, and none of the chloroplast
genes psbA, psbD and rbcL wasreportedtobesignificantly
affected in its expression by this mutation [50]. This suggests
that the lack of one particular sigma factor may have less
deleterious effects than the converse situation, where a factor
physically interferes with transcription unless it is converted
into its active form. Proteolytic cleavage (Fig. 8) is just one
of a number of possible mechanisms to achieve this,
considering the widespread occurrence of protein modifica-
tions that can affect the activity of transcription factors [51].
To address the question as to what extent the SASIG
factors reveal selective promoter affinity, we carried out
Fig. 8. Scheme depicting the possible in vivo control of plastid tran-
scription by proteolytic cleavage of the SASIG3 factor. (A) The full-size
SASIG3 Ôpro-factorÕ is capable of binding to the psbA promoter but
does not allow efficient transcription. (B) The SASIG3 motif with
similarity to N-terminal residues of the r
K
pro-factor from B. subtilis
(inset). It is suggested that upon proteolytic cleavage the mature
SASIG3 protein might confer the ability of transcription initiation on

the RNA polymerase complex. Note that, as a result of similarities in
DNA binding affinity of the multiple SASIG proteins, the transcrip-
tion driven by SASIG1 or SASIG2 can likewise be affected by the
binding of (in)active SASIG3. (C) Alignment of the pro-sequence of r
K
from B. subtilis with SASIG3. Identical amino acids are marked by
asterisks and conserved substitutions by dots.
Ó FEBS 2003 Recombinant plastid sigma factors (Eur. J. Biochem. 270) 1297
competition EMSA experiments using several different
chloroplast promoters (Fig. 5). The strong psbA promoter
is recognized by all SASIG factors, and with comparable
affinity by each factor. Although the other plastid promo-
ters tested in Fig. 5 acted more weakly in terms of competitor
strength compared to the psbA promoter. In a relatively few
instances there were noticeable differences in the promoter
interaction of the individual plastid sigma factors e.g. in the
case of the rbcL vs. ycf3 promoters (Fig. 5). This supports
the view that, rather than acting in a strictly promoter-
selective way, the multiple plastid sigma factors seem to be
capable of substituting for each other in vivo.
Development and environmental cues are likely to play a
role in the expression patterns of the SASIG factors, as is
indicated, for example, by the complementary mode of light
vs. dark accumulation of SASIG2 and SASIG3 (Fig. 2B).
ATSIG5, one of the Arabidopsis factors, has been reported
to reveal a blue-light activated mode of gene expression [41].
Changes in sigma factor abundance and/or activity reflect
the activity of interacting proteins, some of which have
recently been described to play a role in the regulation of
plastid transcription [52–55].

More detailed insight into the determinants for sigma
factor–psbA promoter interactions was sought in the
mutational studies shown in Figs 6 and 7. These experi-
ments provided evidence that the three plant factors reacted
similarly to the mutations in the )35-region but more
diverse to the other regions of the promoter. Previous work
using r
70
from E. coli had shown that mutations in the
recognition helix of the HTH DNA-binding motif in region
4.2 led to altered interaction with the )35-region [28,56].
The mutation of the third base of the psbA)35-region (M-32
G/A) resulted in complete loss of promoter activity for all
sigma proteins. The amino acid at position 16 of the r
70
HTH motif (R588) is known to interact with this specific
base position [28].
In all three mustard sigma factors there is an HTH motif
containing a conserved Arg within region 4.2 (Figs 1 and
7). Because of this similarity with r
70
we reasoned that
Arg455 in SASIG1, Arg545 in SASIG2 and Arg542 in
SASIG3 are the residues that might interact with the
third base of the )35 element of the mustard psbA
promoter. To address this question we substituted Arg455
of SASIG1 by a His and tested the resulting mutant factor
SASIG1–455R/H for promoter binding activity. The
results (Fig. 7B) showing that the effect of the promoter
down-mutation was compensated by the Arg to His change

in the recombinant factor suggest that Arg455 of SASIG1
is indeed involved in the interaction with the third position
of the )35-region.
Using similar mutational approaches, evidence was
obtained for a functional role of at least two other regions
of the plastid sigma factor(s). One is the region 2.5
equivalent [27] found in SASIG1 and SASIG2 but not in
SASIG3 (Fig. 7). With the M-12 G/T mutant of the psbA
promoter that carried a TG to TT change of the extended
)10-region, there was a high decrease in promoter affinity
for either SASIG1, SASIG2 or r
70
, as was expected from
previous findings with r
70
and bacterial promoter mutants
[57]. For SASIG3, however, the decrease was much smaller,
thus reflecting the lack of a conserved region 2.5 in this
factor (Fig. 7).
Furthermore, evidence based on )10-mutants of the psbA
promoter suggests a functional link with a )10 recognition
region of a cloned plastid sigma factor (Fig. 7). Region 2 of
r
70
forms an amphipathic helix [58], a sequence feature that
is conserved in each of the plastid sigma factors (data not
shown). This implies that the general )10 DNA-contacting
mechanism might be comparable and this view has been
strengthened by mutational studies. Conversion of the Glu
at position 437 (region 2.4) of r

70
into a His had previously
been shown to enhance the activity with a mutant promoter
that carried a substitution at the first position of the )10
hexamer [26]. As is evident from Fig. 7A, a conserved Glu is
present in SASIG1 (Q300) and SASIG2 (Q394) but not in
SASIG3, in agreement with the findings (Fig. 6) that
SASIG3 reacted more weakly with the psbA mutant
promoter M-10 G/T. These data suggested that Q300 of
SASIG1 and Q394 of SASIG2 each were involved in the
interaction with the first base of the )10 element of the psbA
promoter. This notion was strengthened by the results
obtained with SASIG1–300Q/H, which showed significant
activity with the mutant promoter (Fig. 7B).
In E. coli, a second residue within region 2.4 of r
70
(T440)
is known to interact with the first base of the )10-hexamer
[56]. As shown in Fig. 7A, none of the three plant factors
contains this conserved Thr, which could reflect differences
in DNA binding between the bacterial and plant sigma
factors.
Acknowledgements
We would like to thank C. Berndt and M. Bo
¨
ckmann for their initial
participation in the cloning of the plastid sigma factors. This work was
funded by the Deutsche Forschungsgemeinschaft (SFB 480-B7).
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