In vitro transcription In vitro transcription reactions 25 lL contained 50 mM Tris/HCl pH 8.0,80 mMNH42SO4,10 mMMgCl2, 1 mM dithiothreitol,600 lMeach of ATP,GTP and CTP,10 lM UTP,20 lCi
Trang 1DNA-binding and transcription characteristics of three cloned sigma
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 rKfrom 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-light-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 (reviewed in [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 AtSig2 and AtSig3 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: gerhard.link@ruhr-uni-bochum.de
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)
Trang 2Isolation of proteins and Western blot analysis
Mustard (S alba) seedlings were grown at 25C for 4 days
either under continuous light (250 lmol photonsÆm)2Æs)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 TaqI covers
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 DdeI and EcoRV; 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
32P-labelled H120 fragment and 0.5 lg E.coli core RNA polymerase in 50 lL of 30 mM Tris/HCl pH 7.0,5 mM
b-mercaptoethanol,0.5 mM EDTA,5% (v/v) glycerol for
10 min at 25C DNA–protein complexes were analysed on
a native 5% (w/v) polyacrylamide gel (29 : 1 acrylamide/ bisacrylamide) containing 0.5MTris/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)
trnK 450 EcoRI/HindIII 352 trnQ 213 EcoRI/TaqI 56 rps16 188 HinfI 107 rbcL 500 BamHI/HindIII 164 ycf3 273 DdeI/EcoRV 158 rrn16 205 DdeI/TaqI 140
Trang 3expressed 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 mM
Tris/HCl pH 8.0,80 mM(NH4)2SO4,10 mMMgCl2, 1 mM
dithiothreitol,600 lMeach of ATP,GTP and CTP,10 lM
UTP,20 lCi [a-32P]UTP (Amersham,400 lCi mmol)1),
10 U RNaseOUT (BRL),0.05 U of E.coli RNA poly-merase holoenzyme (Roche) or 25 nM core enzyme (Epi-centre),and 1 lg double-stranded EcoRI-linearized DNA (pSA05/H120 carrying the psbA promoter; see Table 1 and section Cloned Fragments above) Sigma proteins were added to give a final concentration of 100 nM Following preincubation at 30C 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 fromS 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 r70-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 alba L) (A) Upper: Principal regions of eubacterial r70-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 r70family 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.
Trang 4possible (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 ofCHLOROP [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 reactranscrip-tion 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).
Trang 5were capable of faithful and efficient binding and
transcrip-tion initiatranscrip-tion 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 (b position) as compared to
the free probe (f 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 psbApromoter 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 (f) and of protein-DNA complexes (b) are given in the right margin.
Trang 6For 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 r70from 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 r70,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 psbApromoter 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 and SASIG2 compared to SASIG3 and r70 This was most 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
r70seemed 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 r70),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.
Trang 7which 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 psbApromoter 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 r70 (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)
In the case of r70the 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 r70the 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 related to that of bacterial r70
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.
Trang 8unmodified 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.
Trang 9In 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 vitrotranscription experiments (Fig 4) In the gel shift assays each of the three proteins was found to interact with E.colicore 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 r70known 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.
Trang 10to the situation for r70,which upon cleavage of its N
terminus becomes able to bind to promoter DNA in the
absence of core enzyme [30] Furthermore,in native r70
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 rKfrom B.subtilis [45] (Fig 8C) It has long
been known that certain sporulation factors (rE, rK) 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 pro 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 vivoas 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 was reported to be significantly 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 pro-factor 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.