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J Biol Chem, Vol. 273, Issue 46, 30762-30769, November 13, 1998
From the Expression of light harvesting II genes and of
bacteriochlorophyll and carotenoid biosynthesis genes in
Rhodobacter capsulatus is repressed under aerobic growth
conditions by the transcription factor CrtJ. In this study, we
demonstrate that the crtA-crtI intergenic region contains
divergent promoters that initiate transcription 116 base pairs apart,
based on primer extension analyses. DNase I protection assays
demonstrate that purified CrtJ binds to one palindrome that overlaps
the crtA Like most species of anoxygenic (non-oxygen evolving)
photosynthetic bacteria, Rhodobacter capsulatus represses
synthesis of its photosynthetic apparatus under aerobic growth
conditions (1). Oxygen repression involves the regulation of
photosynthesis gene expression, which is controlled by overlapping
regulatory circuits (reviewed in Ref. 2). Anaerobic activation requires a two-component system composed of a sensor histidine kinase (RegB) and
a response regulator (RegA) to activate reaction center, light harvesting I, and light harvesting II structural genes that are encoded
by the puh, puf, and puc operons, respectively
(3-5). The aerobic repression circuit uses the repressor CrtJ to
decrease aerobic expression of bacteriochlorophyll (bch) and
carotenoid (crt) biosynthesis genes and of light harvesting
II structural genes (puc) (6).
CrtJ-repressed promoters typically exhibit an Escherichia
coli-like In addition to promoters that have two closely spaced palindromes,
there are several other CrtJ regulated promoters that appear to contain
only one palindrome in the In this study, we used DNase I footprint analysis, gel mobility shift,
and filter binding assays to demonstrate cooperative binding of CrtJ to
the two separated palindromes in the puc and crtA-crtI promoter regions. We also demonstrated that
cooperative interactions are required for efficient binding of CrtJ
in vitro and for efficient aerobic repression in
vivo. The mechanism of cooperativity between distant sites will be discussed.
Bacterial Strains, Media, and Culture Conditions--
The
wild-type R. capsulatus strain SB1003 (20) and the
crtJ-disrupted strain DB469 (21) were grown at 34 °C in PYS medium (22). Cells were grown under aerobic/dark or anaerobic photosynthetic conditions as described previously (22). E. coli strains
were grown aerobically in Luria-Bertani medium at 37 °C
(23). Antibiotics were added at the following concentrations:
ampicillin, 100 mg/liter; spectinomycin, 100 mg/liter; and
tetracycline, 10 mg/liter for E. coli; and kanamycin, 10 mg/liter; spectinomycin, 10 mg/liter; and tetracycline, 1 mg/liter for
R. capsulatus.
Cloning and Plasmid Mobilization--
For the crtI
expression vector, a 2.7-kilobase SmaI-HindIII
restriction fragment from pCrtI:Z (6) was cloned into the vector pPHU235 (24) cut by ScaI-HindIII, creating the
vector pES2. Amplification of a fragment containing only the
crtI palindrome was performed by polymerase chain reaction
(PCR) using pES2 as template, and using primers Ose25, 5'-CTC
GAG TCT GGG TCC CTT GTA AT (which introduced a XhoI
site, indicated in boldface letters, in the amplified fragment), and
Ose26, 5'-AGC AAG CTT GGC TGC AGG TCG. The 1.5-kilobase
amplified fragment was purified by using a QIAEX II gel extraction kit
(Qiagen), and cloned into the pCR-Script SK(+) plasmid (Stratagene),
creating the plasmid pES41. A 1.5-kilobase
XhoI-HindIII fragment from pES41 was then
subcloned into XhoI-HindIII sites in pPHU235 to
construct the plasmid pES42. Plasmids pDN12S and pDN13S, which contain
defined puc:lacZ fusions (16), and pPHU235-derived plasmids
were mated into recipient strains as described by Nickens and Bauer
(16).
Overexpression and Purification of CrtJ--
CrtJ fused to an
N-terminal tag of six histidine residues was overproduced using
BL21(DE3)/pET28::CrtJ and purified as described by Ponnampalam and
Bauer (10). To reduce the amount of inclusion bodies formed, the cells
were grown at 34 °C with a slow shaking (250 rpm) and induced when
cultures reached an A600 of 0.9, by the addition
of isopropyl- RNA Isolation and Primer Extension--
Total RNA was isolated
from a 600 ml culture of R. capsulatus SB1003 cells grown
photosynthetically in PYS medium using the guanidinium
thiocyanate-cesium chloride method of Kocabiyik (28) with modifications
described by Kouadio (29).
For primer extension, the primers CrtA-F (5'-CGTCGAAACGGAACAGGCTGA) and
CrtI-R (5'-ACAACGGCGCGACCCATACC) were commercially synthesized;
these primers are designed to anneal the crtA and crtI transcripts, respectively. 32P labeling of
the primers was performed as described by Jiang and Bauer (30). Primer
extension was carried out with 10 µg of total RNA per reaction, as
previously reported by Kouadio (29). A dideoxynucleotide sequencing
ladder was obtained as described by the manufacturer (Sequenase
sequencing kit, United States Biochemical) using the same labeled
primers and the plasmid pCrtI:Z as a template (6). Prior to loading
onto a 6% urea denaturing polyacrylamide gel, the reactions were
heated at 90 °C for 5 min.
Gel Mobility Shift Assays--
Three probes were used for
studying the effect of cooperative binding of CrtJ to the
pucB or crtA/crtI promoter regions. Probes were
prepared by PCR amplification using oligonucleotide primers that were
5'-end-labeled with 32P as described above. The amplified
DNA segments were then purified by electrophoresis in a
nondenaturating 5% polyacrylamide gel and recovered by electroelution.
Amplification of the upstream (
Gel mobility assays were performed by first preparing 5 µl of 4×
binding buffer (composed of 40 mM Tris-HCl (pH 8.0), 200 mM potassium acetate, 4 mM DTT, 5 µl of
dH2O, and 6 µl of different dilutions of purified CrtJ)
to which 4 µl of the DNA substrate were added (composed of 4 fmol of
32P-end labeled DNA probe and heparin as a nonspecific
competitor at a 500-fold weight excess relative to the probe). Samples
were incubated for 30 min at room temperature, loaded on a native 4% Tris-glycine-EDTA-buffered polyacrylamide gel, and electrophoresed at
room temperature. The polyacrylamide gel was then dried and autoradiographed overnight at DNase I Footprint Analysis--
PCR-amplified DNA segments of
the puc promoter and of the crtA-crtI intergenic
region that contained both palindromes, as described for the gel
mobility shift assays, were used for DNase I footprint analysis. For
selective labeling of DNA strands, one of the primers in the PCR was 5'
32P-end-labeled prior to amplification. A 10-µl binding
reaction mixture was first prepared containing 1 µl of DNA (22 fmol),
7 µl of H2O, and 2 µl of 5× footprint binding buffer
composed of 125 mM Hepes (pH 8.0), 250 mM
potassium acetate, 25 mM magnesium acetate, 10 mM calcium chloride, 5 mM DTT, and 125 µg/ml
bovine serum albumin. The reaction mixture was then added to a 10-µl solution of 1× footprint binding buffer containing various amounts of
CrtJ. Digestion with DNase I and subsequent termination of the assay
were then carried out as described previously (31). A modified Maxam
and Gilbert G + A chemical sequencing reaction was used for determining
the location of DNase I protection (32).
Filter Binding Assays--
Nitrocellulose filter binding assays
were used to determine the fraction of purified CrtJ that was active in
DNA binding (10, 33) as well as to determine the effective
concentration for 50% response (EC50). For both assays,
the binding reaction conditions were performed with the same volume and
composition as that used for the gel mobility shift assays, with the
exception of the absence of competitor and the addition of 0.1%
Nonidet P-40 and 0.5 mg/ml bovine serum albumin in the CrtJ dilution
buffer. The binding assays involved a 30-min incubation of the 20-µl
reaction mixtures at room temperature followed by filtering the mixture
through 0.45-µm pore size nitrocellulose filters (BA85, Schleicher & Schuell) that had been presoaked in 1× binding buffer for at least
1 h at room temperature. Filters were then washed once with 1 ml
of 1× binding buffer, air-dried in scintillation vials, and counted in
a Beckman LS 6500 scintillation counter in presence of 1 ml of Bio-Safe
II biodegradable counting mixture (Research Products International
Corp.).
Nitrocellulose-filter-retention efficiencies, defined as the fraction
of the total protein-DNA complexes retained on the filter, were
calculated with 0.2-1 fmol of 32P-labeled DNA probe and
varying amounts of CrtJ (from 0.02 to 90 pmol), and they ranged from 78 to 100%. To determine the percent of active protein, DNA excess assays
contained 0.6 or 1 pmol of purified CrtJ incubated with varying amounts
(from 0 to 1 pmol) of 32P-labeled DNA probes of the
puc promoter, containing either one or the two palindromes.
The percentage of active protein was then calculated from the retention
efficiencies of the filter and number of CrtJ binding sites as
described in Ponnampalam and Bauer (10). CrtJ purified using the
modified expression system described above was typically 8% active.
For EC50 calculation, the CrtJ DNA binding curves were
drawn using curve fit values based on the ligand binding equation Cap *
Ln(Kd + Ln),
with the GraFit program, version 3.03 (Erithacus Software Ltd.).
Determination of Transcription Initiation Sites in the crtA-crtI
Intergenic Region--
We undertook an analysis of transcription start
sites in the crtA-crtI intergenic region as a prelude to
analysis of CrtJ binding to this region. Primer extension analysis
using a crtI specific primer yielded a major product that
corresponds to a G residue located 18 bp upstream of the start codon of
crtI and two minor products located 9 and 10 nucleotides
upstream (Fig. 1A). Inspection
of the sequence immediately upstream of the major primer extension
product revealed a putative
Primer extension analysis of crtA gave rise to a major
product with a 5'-end that corresponds to a transcription start site located 23 bp upstream of the crtA translation start codon,
as well as minor products located 75-77 bp upstream (Fig.
1B). The region upstream from the major product contains
CrtJ Binds to Distant Palindromes in the crtA-crtI Intergenic
Region and in the puc Promoter Region--
DNase I protection assays
were performed on both strands of the crtA-crtI
promoter region to determine whether CrtJ binds to the palindromes that
were identified by sequence analysis. The DNase I digestion patterns in
Fig. 3 show that CrtJ protects the
palindrome that spans the
We also undertook an analysis of CrtJ binding to the puc
promoter using similar techniques. Inspection of the sequence of this
promoter region (Fig. 2B) revealed the presence of two
putative CrtJ palindromes, with one adjacent to the puc CrtJ Binds Cooperatively to the crtA, crtI, and puc
Palindromes--
We next addressed whether cooperative interactions
may occur among CrtJ bound to the palindrome sites located in the
crtA and crtI promoters or to the distantly
separated puc binding sites, using gel retardation assays
with probes that contained one or both of the relevant palindromes. As
shown in Fig. 4A, lanes 1-4, a complete shift of a DNA probe that contained both the crtA
and crtI palindromes was observed with as little as 0.5 µg
of CrtJ. In contrast, a DNA probe containing only the
crtA palindrome (Fig. 4A, lanes 5-8) or only the
crtI palindrome (lanes 9-12) required 2 µg of
CrtJ to bind all the probe. (Note that the crtA and the crtI probes (Fig. 4A, lanes 5-12) are
contaminated with another DNA fragment, amplified during the PCR step
and co-purified with the specific probes. The absence of a shift with
these contaminating DNA fragments indicates good specificity for CrtJ
binding to the target DNA).
Similar gel retardation assays were performed with DNA probes made from
the puc promoter region. Using a probe containing both
palindromes, we observed a complete shift with the lowest amount (0.35 µg) of CrtJ used (Fig. 4B, lanes 1-4). When more CrtJ was
added, the shifted fragment exhibited even slower mobility, presumably
caused by nonspecific binding of CrtJ or by the formation of highly
ordered DNA-protein complexes (Fig. 4B, lanes 3 and 4). When CrtJ was incubated with probes containing only the
upstream (Fig. 4B, lanes 5-8) or downstream (Fig. 4B,
lanes 9-12) palindrome, a shift of the entire probe required 1.4 µg of purified CrtJ.
To measure CrtJ-DNA binding affinities, we performed nitrocellulose
filter binding assays with probes that contained one or both of the
palindromic sites in the crtA-crtI and puc
promoter regions. For this analysis, the same probes that were used for the gel mobility shift assays were analyzed for filter retention with
various amounts of CrtJ. The resulting binding curves (Fig. 5A and B) indicate
that probes that contained single palindromes produced EC50
values 6-10-fold higher than for probes that contained two
palindromes. When corrected for the percentage of active protein (see
under "Materials and Methods"), the calculated EC50
value for a probe with both the crtA and crtI
palindromes was 2.6 × 10 The in Vivo Requirement for Two Distant Palindromes for Activity of
the Repressor CrtJ--
The in vivo requirement for two
distant palindromes for CrtJ-mediated repression of gene expression was
studied using lacZ reporter fusions in R. capsulatus. For analysis of puc expression, we used
plasmid pDN13S, which has an extended 1051-bp puc promoter segment containing both palindromes, and plasmid pDN12S, which has a
truncated 284-bp puc promoter segment containing only the downstream (
Two crtI:lacZ fusions were also constructed to assess the
requirement for distantly separated CrtJ-binding sites in the
crtA-crtI intergenic region. Plasmid pES2 contains the
entire crtA-crtI intergenic region, including both the
crtA and the crtI palindromes, whereas plasmid
pES42 carries a truncated intergenic region in which the
crtA palindrome had been deleted (Fig. 7C). As
observed in Fig. 7D, the wild-type cells harboring the
construct containing only the crtI palindrome (pES42) had
elevated aerobic
Thus, we conclude that the two CrtJ palindromes separated by 240 bp in
the puc promoter region and by 76 bp in the
crtA-crtI intergenic region are required for efficient
CrtJ-mediated repression of these genes in vivo.
This study demonstrates that CrtJ binds to two palindromes that
are located 76 bp apart in the crtA-crtI intergenic region and to two palindromes located 240 bp apart in the puc
promoter region. Efficient CrtJ repression of crtA, crtI,
and puc expression involves cooperative interactions between
CrtJ bound to these palindromes. Cooperative interaction has also been
demonstrated for CrtJ repression of the bchC promoter, which
has two closely spaced (neighboring) palindromes (10, 11). Thus, a
common characteristic of DNA binding by CrtJ appears to be a
requirement for two binding sites that are either closely spaced, as
for the bchC promoter, or distant, as for the puc
and the crtA and crtI promoters.
Quantitative filter binding analyses indicate that CrtJ has similar
affinities for the individual binding sites that are in the puc,
crtA and crtI promoters, as demonstrated by
EC50 values that range from 1.6 × 10 Compared with individual sites, the affinity of CrtJ for DNA fragments
that contain the two puc palindromes, or both the
crtA and crtI palindromes, is 6-11-fold higher
(3.2 × 10 Our analysis of transcription start sites in the crtA-crtI
intergenic region indicates that one of the CrtJ-binding sites overlaps
a Disruption of crtJ caused increased aerobic expression of
bch and crt genes, but a residual level of
anaerobic activation was still observed, suggesting that these genes
are subject to aerobic repression by CrtJ and anaerobic activation by
unknown factors (inducers) (6, 12). Similarly, expression of the puc operon is repressed by CrtJ under aerobic growth
conditions and induced under anaerobic growth conditions by the
two-component regulatory proteins, RegB and RegA (4-6, 9). Recently,
Du et al. (41) isolated a mutant version of RegA, called
RegA*, and demonstrated that RegA* binds to the puc
promoter, protecting two regions extending from As indicated above, cooperative interactions between CrtJ bound to
widely separated sites on the DNA presumably involves the formation of
a DNA loop (Fig. 8). Protein-mediated
looped complexes are important regulatory elements in many systems.
Examples include the regulation of prokaryotic and eukaryotic gene
expression, site-specific recombination, and DNA replication (reviewed
in Ref. 42). Different factors are known to facilitate the formation and stability of DNA-protein looped complexes. This includes the presence of intrinsically bent DNA sequences or the binding of proteins
that bend DNA. For instance, integration host factor (IHF) (43), HU
(44), and cAMP receptor protein (45, 46) can stabilize protein-mediated
looped complexes.
There is no direct evidence that cooperative binding by CrtJ to distant
sites occurs via a DNA loop. However, there is one observation that
favors a DNA looping in CrtJ-mediated repression of gene expression.
Indeed IHF, which is a sequence-specific DNA-binding protein that
induces a severe bend (>160°) in the DNA helix (47), has recently
been shown to bind between the two CrtJ palindromes on the
puc promoter (16). Sequence scanning has revealed putative IHF-binding sites in the crtA-crtI intergenic sequence (data
not shown). Thus CrtJ-induced looped DNA could be stabilized by IHF in vivo. The possible involvement of IHF in the mechanism of
CrtJ-mediated repression is currently under investigation.
We thank Terry H. Bird for careful reading of
the manuscript.
*
This work was supported by National Institutes of Health
Grants GM53940 and GM00618 (to C. E. B).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.
¶
Present address: Dept. of Cell Biology, The Cleveland Clinic
Foundation, 9500 Euclid Ave., Cleveland, OH 44195.
The abbreviations used are:
bp, base pair(s); PCR, polymerase chain reaction; IHF, integration host factor.
CrtJ Bound to Distant Binding Sites Interacts Cooperatively to
Aerobically Repress Photopigment Biosynthesis and Light Harvesting II
Gene Expression in Rhodobacter capsulatus*
,
Department of Biology and the
§ Biochemistry Program, Department of Chemistry, Indiana
University, Bloomington, Indiana 47405
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
10 promoter recognition sequence as well as to a
second palindrome that overlaps the 35 crtI promoter recognition sequence. Similar analyses also show that the
puc promoter region contains two distant CrtJ palindromes,
with one near the 35 promoter recognition sequence and the other
located 240 base pairs upstream. Gel mobility shift and filter
retention assays indicate that CrtJ binds in a cooperative manner to
these distantly separated palindromes. In vivo expression
assays with puc and crtI promoter reporter
plasmids further demonstrate that aerobic repression of puc
and crtI expression requires both CrtJ palindromes. These
in vitro and in vivo results indicate that aerobic repression of puc, crtA, and crtI
expression involves cooperative interactions between CrtJ bound to
distant palindromes. A DNA looping model is discussed.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-70 sequence motif with one or two copies of a
conserved CrtJ recognition palindrome TGTN12ACA in the 10
or 35 region (7-9). For example, the R. capsulatus bchC
promoter has two CrtJ palindromes spaced 8 bp1 apart, with one spanning
the 10 and the other the 35 promoter recognition sequences. Binding
of CrtJ to these sites is redox-dependent, with in
vitro binding much tighter under oxidizing conditions than under
reducing conditions (10). As shown in a companion study by Ponnampalam
et al. (11), repression of bchC involves cooperative interactions between CrtJ bound to adjacent palindromes. Consequently, mutations in either palindrome disrupt in vivo
regulation by CrtJ (12). The spacing between the two bchC
palindromes is also critical, as demonstrated by the observation that
+6- and +11-bp additions between the palindromes abolished CrtJ binding (11). A similar arrangement of CrtJ palindromes spanning the 35 and
the 10 promoter regions is also found in the Rhodobacter sphaeroides bchC and puc promoters (13, 14) and in the
Rhodopseudomonas palustris pucB and pucE
promoters (15). This suggests that cooperative interactions between
CrtJ bound to adjacent palindromes may be a highly conserved mechanism
of repression.
10 to 40 region. For example, the
intergenic sequence between the divergently transcribed crtI and crtA genes from R. capsulatus contains two
palindromes located 76 bp apart (distance calculated from the axis of
dyad symmetry for each palindrome). One palindrome overlaps a potential
10 region of the crtA promoter, and the other overlaps a
potential 35 region for the crtI promoter (8). Inspection
of the puc promoter region also indicates the presence of a
single palindrome near the 35 promoter recognition sequence (16),
with a second putative CrtJ palindrome located 240 bp upstream. The
presence of two distant CrtJ palindromes in the puc
promoter, and in the crtA-crtI intergenic region, is
reminiscent of the gal, ara, or lac
systems, which require multiple binding sites to obtain full repression
of gene expression (17-19). This raises the possibility that binding
of CrtJ to distantly separated palindromes may be a requirement for
full repressive activity of CrtJ in this second class of promoters.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-Galactosidase Assays--
-Galactosidase activity was
assayed from the cell culture as described by Miller (25) and modified
as described by Elsen et al. (26).
-D-thiogalactopyranoside to a final concentration of 1 mM. The purified protein was partitioned
into 10-µl aliquots and stored at 80 °C. The final protein
concentration, which was typically 1.6 mg/ml, was measured with the
CrtJ extinction coefficient (
180), which was determined using the
A205/A280 method described by Scopes (27).
279 to 296 bp) puc
palindrome utilized primers PucB-F3 (5'-CAATTCGACCTGAAAATTCC) and
PucB-R2 (5'-CATCTTTGGGCGATTCCGGG), whereas amplification of the
downstream palindrome (39 to 56 bp) used primers PucB-F
(5'-CCCGGAATCGCCCAAAGATG) and PucB-R3 (5'-AAGCTCTCTCGTTAAGTTC).
For amplification of both upstream and downstream palindromes,
primers PucB-F3 and PucB-R3 were utilized. Plasmid pLHIIZ (5) was first
digested with EcoRI prior to use as a template for
amplification of the puc promoter region. For amplification
of the crtA palindrome, the primers CrtA-F and CrtI-R2
(5'-TGTCTCGCGACAAAACGTCTG) were used. Amplification of the
crtI palindrome used primers CrtA-F3
(5'-CTCAGACGTTTTGTCGCGAGACA) and CrtI-R. Amplification of both
palindromes in the crtA-crtI intergenic region used primers
CrtA-F and CrtI-R. The plasmid pCrtI:Z (6), which was used as a
template for crtA-crtI promoter amplification,
was digested with BamHI prior to amplification.
80 °C with an intensifying screen.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-70 type promoter on the basis of
sequences located 10 (AATGCA) and 35 (TTGACG) bp upstream (Fig.
2A). As indicated in Fig. 2,
this region contains a single CrtJ binding sequence
(TGTN12ACG) that overlaps the 35 promoter motif. Putative
10 (TATCAT) and 35 (TTGTAA) sequences can also be identified
upstream from the weak start sites, with the CrtJ binding site
overlapping the 35 region.
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Fig. 1.
Primer extension analysis of transcripts
initiating from the crtA-crtI intergenic region.
A and B show primer extension products initiated
from the crtI and crtA promoters, respectively.
The A, C, G, and T lanes represent dideoxynucleotide sequencing ladders
generated with the same primers that were used for primer extension.
Nucleotides corresponding to the primer extension bands are indicated
on the printed sequence by arrows on the right.
Boxed bases are the potential 70 10 and
35 promoter consensus sequences, with the two pairs of
half-arrows representing CrtJ-recognition palindromes.
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Fig. 2.
Features of the crtA-crtI and
puc promoter sequences. A and B
are DNA sequences of the crtA-crtI intergenic and
puc promoter regions, respectively. White letters
on a black background with arrows denote the
CrtJ-palindromic sites. Single bars below or above the DNA
sequences indicate potential 35 and 10 promoter sequences.
Large arrows indicate the start site and direction of
transcription of each gene, whereas smaller arrows indicate
additional minor start sites. Regions protected from DNase I digestion
by CrtJ binding are highlighted in gray. The beginning of
peptide sequences for each gene is also indicated.
10 (AATATC) and 35 (ATTACA) promoter recognition sequences and a
potential CrtJ binding palindrome (TGTN12ACA) that overlaps
the 10 recognition sequence (Fig. 2A). Inspection of the
sequence upstream from the minor products did not reveal obvious 10
or 35 promoter recognition sequences (Fig. 2A). If the
upstream primer extension products do represent a transcription start
site, this second crtA promoter is likely to be regulated by
CrtJ because the CrtJ palindrome that spans the crtI
promoter also overlaps this promoter (Fig. 2A).
10 region of the crtA promoter (best resolved with the top strand in Fig. 3A), as well as
the palindrome that spans the 35 region of the crtI
promoter (best resolved in Fig. 3B). Several DNase I
hypersensitive sites are present in each of the protected regions.
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Fig. 3.
DNase I footprint analysis of CrtJ binding to
the crtA-crtI intergenic sequence and to the puc
promoter. A, CrtJ-mediated DNase I protection
patterns to the top strand of the crtA-crtI
intergenic region, which shows protection to the crtA
promoter region. B, DNase I protection patterns to the
bottom strand of the crtA-crtI intergenic region,
which shows protection to the crtI promoter region.
C and D are CrtJ protection patterns to the top
and bottom strands of the puc promoter, respectively.
G + A indicates a Maxam and Gilbert sequencing ladder. The
second lane of each gel (0) is a DNase I-digested probe
incubated in the absence of CrtJ. Each of the subsequent lanes are
protection patterns generated in the presence of increasing
concentrations (in µM) of purified CrtJ. The
arrows at +1 represent the start and direction of
transcription, and the vertical arrows on the
right of each panel represent the CrtJ palindromic
sequences, as indicated in Fig. 2.
35
motif (downstream palindrome) and the other one located 240 bp upstream
(upstream palindrome). As showed by the DNase I digestion pattern
generated with the top strand (Fig. 3C), CrtJ protects a
region extending from 274 to 308 bp that corresponds to the
upstream CrtJ palindrome. DNase I protection patterns generated with
the bottom strand (Fig. 3D) exhibit CrtJ protection
extending from 37 to 62 bp that corresponds to the downstream CrtJ
palindrome. Several hypersensitive sites to DNase I digestion are also
observed within these protected regions (Fig. 3, C and
D).
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Fig. 4.
Cooperative binding of CrtJ to the
crtA-crtI intergenic sequence and to the puc
promoter as assayed by gel retardation assays. A,
gel mobility shift of CrtJ binding to the crtA-crtI
intergenic region. Reactions used probes of 232 bp containing both
palindromes (lanes 1-4), 122 bp containing only the
crtA palindrome (lanes 5-8), or 133 bp
containing only the crtI palindrome (lanes
9-12). Four fmol of each 32P-labeled DNA probe were
incubated with various amounts (0, 0.5, 1, or 2 µg) of purified CrtJ.
B, gel mobility shift of CrtJ binding to the puc
promoter region. Reactions used probes of 482 bp containing both
palindromes (lanes 1-4), 282 bp containing only the
upstream palindrome (lanes 5-8), or 220 bp containing only
the downstream palindrome (lanes 9-12). The amounts of
purified CrtJ used for each set of reactions were 0, 0.35, 0.7, and 1.4 µg. The dots in lanes 1, 5, and 9 distinguish specific probes from contaminating fragments.
9 M,
versus 3.2 × 108 and 2.4 × 108 M for probes that contained only the
crtA or crtI palindromes, respectively. For
experiments that measured CrtJ binding to the puc promoter
region, the calculated EC50 value for the probe containing both palindromes was 3.2 × 109 M,
compared with 1.6 × 108 and 2 × 108 M for probes that contained only the
upstream (279 to 296) or downstream (39 to 56) palindromes,
respectively. Fig. 6 summarizes the
observed EC50 values for the various individual
binding sites that were analyzed in this study, as well as the observed
EC50 value for the bchC upstream palindrome that
is reported in the companion study by Ponnampalam et al.
(11).
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Fig. 5.
CrtJ protein excess binding curves.
Filter binding assays involving CrtJ and the crtA-I
intergenic sequence (A) or the puc promoter
region (B) utilized the same DNA fragments that were used
for gel retardation assays. Individual assays contained 1 fmol of DNA
probes from the crtA-crtI region or 0.2 fmol of the
puc promoter region. Binding curves were obtained with
probes containing both palindromes (closed circles), either
the crtA promoter or puc upstream palindrome
(open circles), or the crtI promoter or
puc downstream palindrome (open triangles).
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Fig. 6.
Sequence alignment of the palindromic sites
protected from DNase I digestion by CrtJ binding that are present in
the bchC, puc, crtA, and crtI
promoters. EC50 values for CrtJ binding to each
palindrome are listed on the right. In the consensus
sequence, lowercase letters indicate four bases in common,
uppercase letters indicate five shared bases, and
boldface uppercase letters indicate strictly conserved
bases. ND, not determined.
39 to 56) palindrome (Fig.
7A) (16). When assayed for
-galactosidase activity in aerobically grown wild-type R. capsulatus cells (SB1003), we observed that cells harboring the construct with only one palindrome (pDN12S) have a significantly higher
level of activity (144%) over cells that contain the construct that
has two palindromes (pDN13S) (Fig. 7B). This elevated
activity observed with pDN12S is clearly a result of the inability of
CrtJ to effectively repress puc expression, as the level of
activity is similar to that observed with the
crtJ-disrupted strain DB469 carrying either
pDN12S or pDN13S (Fig. 7B).
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Fig. 7.
In vivo role of the CrtJ palindromes in
the puc and crtI promoters. A
and C depict plasmid constructs that contain one or two CrtJ
palindromes and were used to assay for puc and
crtI expression, respectively. B and D
give results of -galactosidase activity assays from aerobically
grown cells of the wild-type strain, SB1003, and the
crtJ-disrupted strain, DB469. Units of -galactosidase
activity represent µmoles of
o-nitrophenyl--D-galactopyranoside
hydrolyzed/min/mg of protein. Error bars denote standard
deviation.
-galactosidase activity (133%) over cells that
contained the plasmid that has both palindromes (pES2). This reflects
the impairment of CrtJ to repress crtI expression if only
one palindrome is present. Furthermore, when the crtA
palindrome was deleted, no regulation of crtI expression by
CrtJ was retained, as evidenced by similar -galactosidase activities
in SB1003 and the crtJ-disrupted strain DB469 carrying
plasmid pES42 (Fig. 7D).
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
8
to 3.2 × 108 M. Surprisingly, CrtJ has
the same affinity for the TGTN12ACA palindromes as for the
variant TGTN12ACG that is present in the crtI promoter (Fig. 6). These values are very similar to
that reported for the upstream bchC palindrome (7.8 × 108 M) by Ponnampalam et al. (11)
in the companion study. In contrast, CrtJ exhibits a significantly
reduced affinity (>1 µM) for the downstream
bchC palindrome (11) despite the fact that this palindrome still exhibits a very good consensus sequence (Fig. 6). The only significant difference is that this sequence exhibits a substitution of
an A for a T two nucleotides upstream from the ACA that is conserved in
the other palindromes. This indicates that a T at this position may be
critical for effective CrtJ binding.
9 and 2.6 × 109
M, respectively). This increase of affinity is similar to
the observed 26-fold increase in CrtJ binding to two palindromes in the
bchC promoter region over a single binding site (11). Even though there must be some fundamental differences in the interactions among CrtJ bound to these two different classes of promoters
(i.e. the potential involvement of DNA looping between
distant palindromes versus direct interaction between
adjacent palindromes), the relative binding affinities and level of
repression are conserved.
-70 type 35 motif for the crtI promoter. The other binding site overlaps the 10 motif for the crtA promoter.
Thus, cooperative binding of CrtJ to these two palindromes provides a
mechanism of coordinately repressing these divergent promoters. What is
the significance of co-repressing the crtA and
crtI promoters? Previous studies have postulated that
crtI is the first of a two-gene operon that also contains
crtB (7, 34). crtI and crtB are known
to code for phytoene dehydrogenase and phytoene synthase, respectively,
which catalyze two of the earliest steps in carotenoid biosynthesis
(35-38). It has also been demonstrated that the translational stop
codon for crtA overlaps the translational start codon for bchI; thus, the transcripts initiating at the
crtA promoter should transcribe the downstream
bchI gene (34, 39). bchI codes for a subunit of
magnesium chelatase, an enzyme involved in inserting Mg2+
into protoporphyrin IX forming the first intermediate in the bacteriochlorophyll branch of the tetrapyrrole biosynthetic pathway (34, 40). Thus, coordinate repression of the crtA and
crtI promoters could influence the flow of metabolic
intermediates into both carotenoid and bacteriochlorophyll biosynthetic pathways.
52 to 69 and 73
to 80. Thus, RegA binds to a region of the puc promoter
that partially overlaps the downstream binding site of CrtJ (from 37
to 62). Consequently, the two proteins may compete in vivo
for their respective binding sites. Furthermore, deletion and
mutational analyses of the bchC promoter region indicate
that this promoter is activated under anaerobic conditions by a
cis-acting site (AT-rich region) located just upstream of
the -70-like sequence of the promoter (12). This region overlaps the
bchC upstream palindrome of CrtJ (10). It is therefore
possible that, like the puc and bchC promoters, other crt and bch genes are controlled by CrtJ
competing with a transcriptional activator for overlapping binding sites.
View larger version (21K):
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Fig. 8.
Model of the cooperative binding of CrtJ to
the crtA-crtI intergenic region. Arrows
above the genes indicate the direction of gene transcription, and the
arrows in the expanded portion below the genes show the
start site and direction of transcription of the crtA and
crtI genes. The two palindromic CrtJ-binding sites are shown
overlapping the 10 region of crtA and the 35 region of
crtI as lines with half-arrows at both
ends. In presence of oxygen, CrtJ dimers bound to either palindromic
site can interact through the formation of a looped DNA structure that
brings the distant sites close together.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biology,
Indiana University, Jordan Hall, Bloomington, IN 47405. Tel.: 812-855-6595; Fax: 812-855-6705; E-mail: cbauer{at}bio.indiana.edu.
REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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