Plant Physiol. (1998) 117: 225-234
Transcriptional and Posttranscriptional Control of mRNA from
lrtA, a Light-Repressed Transcript in
Synechococcus sp. PCC 70021
Hrissi Samartzidou and
William R. Widger*
Department of Biochemical and Biophysical Sciences, University of
Houston, Houston, Texas 77204
 |
ABSTRACT |
Transcription regulation and
transcript stability of a light-repressed transcript,
lrtA, from the cyanobacterium
Synechococcus sp. PCC 7002 were studied using
ribonuclease protection assays. The transcript for lrtA
was not detected in continuously illuminated cells, yet transcript
levels increased when cells were placed in the dark. A lag of 20 to 30 min was seen in the accumulation of this transcript after the cells
were placed in the dark. Transcript synthesis continued in the dark for
3 h and the transcript levels remained elevated for at least
7 h. The addition of 10 µm rifampicin to illuminated
cells before dark adaptation inhibited the transcription of
lrtA in the dark. Upon the addition of rifampicin to 3-h
dark-adapted cells, lrtA transcript levels remained
constant for 30 min and persisted for 3 h. A 3-h half-life was
estimated in the dark, whereas a 4-min half-life was observed in the
light. Extensive secondary structure was predicted for this transcript
within the 5
untranslated region, which is also present in the 5
untranslated region of lrtA from a different
cyanobacterium, Synechocystis sp. PCC 6803. Evidence
suggests that lrtA transcript stability is not the
result of differences in ribonuclease activity from dark to light.
Small amounts of lrtA transcript were detected in
illuminated cells upon the addition of 25 µg mL
1
chloramphenicol. The addition of chloramphenicol to dark-adapted cells
before illumination allowed detection of the lrtA
transcript for longer times in the light relative to controls without
chloramphenicol. These results suggest that lrtA mRNA
processing in the light is different from that in the dark and that
protein synthesis is required for light repression of the
lrtA transcript.
| |
INTRODUCTION |
Unicellular cyanobacteria grown under autotrophic conditions show
a decrease in macromolecular synthesis when cells are placed in the
dark (Singer and Doolittle, 1975
). This is not surprising because the
main energetic process by which cyanobacteria grow, i.e.
photosynthesis, ceases. Cell division and DNA replication have been
reported to stop in the dark (Binder and Chisholm, 1990) and
transcription and translation of most proteins are not detected (Singer
and Doolittle, 1974, 1975; Tan et al., 1994). In
Synechococcus sp. PCC 7002, transcripts encoding proteins
that participate in the photosynthetic machinery, such as
cpcBAC, apcAB, psbA, psbB, petBD, and petCA, are not detectable in
dark-adapted cells, but their levels increase rapidly when the cells
are exposed to light (Brand et al., 1992). Conversely, a
light-repressed transcript, lrtA (Tan et
al., 1994), is transcribed only in the dark. This transcript is not
detected in illuminated cells and is rapidly degraded when dark-adapted
cells are placed in the light. The gene encoding lrtA has
been cloned and sequenced from Synechococcus sp. PCC 7002 (Tan et al., 1994) and is located on a 540-bp open reading frame within
a presumed 1065-nucleotide message. The lrtA transcription
start site is located 380 bp upstream from the translation start and
possesses a consensus Escherichia coli promoter (Tan et al.,
1994). Although the function of the lrtA gene product is
unknown, it has significant sequence similarity to two different proteins. One of these proteins is a unique, chloroplast-specific small-subunit ribosomal protein, S30 (Zhou and Mache, 1989; Johnson et
al., 1990; Schmidt et al., 1993), and the other is a
transcription-modulator protein thought to function in the
two-component bacterial regulatory system (Merrick and Coppard, 1989).
The sequence similarity among these proteins suggests that
lrtA may modulate either transcription and/or translation.
LRTA is one of the first proteins to be synthesized upon illumination
of dark-adapted cells, which further suggests that the lrtA
gene product could regulate others in a timed response.
In this paper we report that the lrtA transcript is more
stable in dark-adapted cells than in cells exposed to light. Also, new
protein synthesis is required for light repression of the lrtA transcript. An extensive 5 secondary structure has
been determined by computer analysis, which could be responsible for the stability of the lrtA message in the cyanobacterium
Synechococcus sp. PCC 7002.
| |
MATERIALS AND METHODS |
Bacterial Growth
Synechococcus sp. PCC 7002, obtained from the American
Type Culture Collection (Rockville, MD), was maintained and cultured under photoautotrophic conditions using an artificial seawater medium
(Widger, 1991). Water-saturated air supplemented with 3 to 5% (v/v)
CO2 was bubbled into 300-mL culture tubes
(42 × 3.5 cm in diameter) exposed to direct, continuous
fluorescent light from two cool-white 30-W bulbs (Sylvania). Tubes were
inoculated with a 5-mL aliquot from a cyanobacterial culture grown in a
50-mL flask started from a single colony grown on agar plates. Cells were grown to early log phase with an optical density of 0.8 to 1.0 at
550 nm. The cells at this concentration were used for the RPAs.
Light and Dark Adaptation
Cells grown under constant light conditions of 20 µE
m2 s1 were considered
to be light adapted, whereas cells placed in total darkness were
considered to be dark adapted. Dark adaptation was for 3 h unless
specified otherwise. Rifampicin was used at a final concentration of 50 µg mL1 and CM was used at 25 µg
mL1. Both inhibitors were incubated for 10 min
before the shift in the light-adaptation regimen.
Recombinant DNA Techniques
Templates for the antisense RNA probe were generated from
the 895-bp BamHI fragment spanning 170 bp upstream of the
lrtA transcription start to 725 bp downstream isolated from
the plasmid pTX100 containing the 2.7-kb EcoRI fragment
encoding the lrtA gene (Tan et al., 1994) (Fig. 1). This
fragment was ligated in the BamHI site of pGEM-3Zf (Promega), creating
pHS
lrtA9B. The fragment orientation in
pHSlrtA9B was determined by double-stranded sequencing
using universal and reverse sequencing primers. pHSlrtA9B
was linearized with EcoRI, and the resulting DNA was used as
a template for in vitro transcription of antisense RNA using the SP6
RNA polymerase. Probes for lrtA, cpcBAC,
petCA, and ndhB were made using the
Maxiscript II kit (Ambion, Austin, TX), according to the
manufacturer's instructions, from 1 µg of DNA.
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| Figure 1.
The putative  35 and 10 elements, the start of
transcription (determined experimentally by the method of Tan et al.,
1994) (vertical arrow), and possible operator sites are shown. The
probe for the RPAs is the 895-bp BamHI fragment (labeled
1) spanning the gene. Other fragments used in the RNase activity
assays, labeled 1 and 2, and the entire mRNA generated from E. coli polymerase are shown. E, EcoRI; B,
BamHI; and H, HindIII.
|
|
The reaction mixture was incubated at room temperature for 2 h,
and 1 µL of DNase I was added and incubated for another 15 min at
37°C. Twenty microliters of gel-loading buffer (80% [v/v] formamide, 0.1% [w/v] xylene cyanol, 0.1% [w/v] bromphenol blue, and 2 mm EDTA) was added and incubated for 5 min at 90°C.
The entire sample was loaded onto a 0.8-mm-thick, 8 m urea,
5% PAGE gel and electrophoresed in 1× Tris-borate-EDTA buffer at 300 V for 1 h. After electrophoresis, gels were exposed to x-ray film for 5 min and the labeled RNA was excised. The excised gel was soaked
in 350 µL of 0.5 m
CH3COONH4, 1 mm
EDTA, and 0.1% (w/v) SDS overnight at 37°C to elute the labeled
probe. An aliquot was taken for liquid-scintillation counting and the
remainder was stored at 20°C. For the synthesis of the full-length
sense lrtA message, Escherichia coli RNA
polymerase isolated by published procedures (Burgess et al., 1975) was
used, taking advantage of the near- E. coli consensus 35
and 10 promoter elements found upstream of the lrtA
transcription start site. A 1.6-kb fragment was amplified by PCR using
primers located at bases 480 and 2040 of the original EcoRI
fragment on pTX100. The RNA polymerase reaction was done on this
fragment in the presence of 150 mm KCl as a modification of
the original transcription protocol described in the Maxiscript II kit.
The full-length lrtA mRNA was gel purified as described above.
RPA
Synechococcus sp. PCC 7002 cells grown from 0.8 to 1.0 A550 as described were incubated in the
light or in the dark. At various times 107 cells
mL1 were harvested by rapid centrifugation and
solubilized in 5 m guanidine thiocyanate and 0.1 m EDTA at room temperature. Cell lysates were stored at
20°C and thawed for hybridization experiments. The cells were
solubilized directly without centrifugation when timed experiments were
done. Aliquots of cells were solubilized by mixing with 2 volumes of 5 m guanidine thiocyanate and 0.1 m EDTA.
Cell lysates (30 µL) were mixed with approximately
106 cpm of probe per reaction (1-3 µL
depending on the specific activity of each probe used). The
hybridization reactions were incubated at 45°C for 16 to 20 h.
After hybridization, the samples were digested with RNase cocktail (1 mg mL1 RNase A and 20,000 units
mL1 RNase T1) for 30 min at 37°C. The samples
were further incubated with 5 µL of 20 mg mL1
proteinase K and 20 µL of 10% (w/v) SDS for an additional 30 min.
After phenol-chloroform extraction and ethanol precipitation, the
recovered nucleic acids were dissolved in gel-loading buffer, incubated
at 95°C for 2 min, and loaded onto a 5% PAGE gel. The samples were
electrophoresed and the positions of the protected RNA fragments were
determined by radiography. Results were quantitated by densitometry
using an EagleEye densitometer (Stratagene). All RPA experiments
were repeated a minimum of three times.
RNase Assays
Protein extracts from light- or dark-adapted cells were checked
for RNase activity. Extracts were isolated as follows: 50 mL of
Synechococcus sp. PCC 7002 cells
(A550 = 1.0) were light or dark adapted as
described and treated with lysozyme (0.5 mg/mL cells) for 20 min. The
resulting spheroplasts were centrifuged and resuspended in 1 mL of
lysis buffer (10% glycerol, 50 mm Tris HCl, 1 mm EDTA, 2 mm DTT, 0.5% [v/v] Triton X-100,
and 1 mm PMSF, pH 7.5) and sonicated at maximum strength
for three 30-s bursts, followed by cooling on ice using a probe
sonicator (model 450, Branson, Danbury, CT). The cell lysates were
centrifuged for 10 min at 10,000 rpm and supernatants were checked for
RNase activities.
Sense or antisense RNA probes were synthesized as described above using
either SP6 or T7 polymerase. Templates for the probes included the
0.89-kb BamHI fragment from lrtA (Fig. 1), the
1.6-kb BamHI fragment from petCA (Brand et al.,
1992), and the 880-bp XhoI-BamHI fragment from
cpcBAC (de Lorimier et al., 1984). The full-length sense
lrtA probe was synthesized using E. coli RNA polymerase.
RNase activity assays were carried out using 10 µg of cell lysate
from either dark- or light-adapted cells, which was mixed with 100,000 cpm of RNA probe in RNase digestion buffer (10 mm Tris-HCl,
pH 7.5, 0.3 m NaCl, 5.0 mm EDTA, and 5 mm MgCl2) in a total volume of 20 µL. Each reaction was incubated at 37°C for various times before
the addition of 100 µL of 0.3 m
NaC2H3O2 and 1 mm EDTA
containing 0.5 µg µL1 yeast RNA. The
samples were immediately extracted with phenol-chloroform and the RNA
was precipitated with ethanol. The recovered nucleic acids were
dissolved in gel-loading buffer and incubated at 95°C for 2 min
before loading onto a 5% polyacrylamide gel. The samples were
subjected to electrophoresis as described above and RNA was visualized
by autoradiography.
| |
RESULTS |
lrtA Transcript Is Synthesized in the Dark but
Not in the Light
The time course for message accumulation in the dark was
determined by RPA using the 895-bp BamHI lrtA
fragment as a probe (Fig. 1). Aliquots
were taken from preilluminated cells placed in the dark at specific
time points (Fig. 2, A and B). The
lrtA transcript was not detected in illuminated cells even
after the radiograms were overexposed. However, the lrtA
transcript was detected 20 to 30 min after the cyanobacterial cells
were placed in the dark, and transcript levels continued to increased
for 3 h. The message levels remained elevated for at least 7 h in the dark. Routinely, a 20- to 30-min lag from the onset of dark treatment to the detection of the transcript was seen.
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| Figure 2.
A, Radiogram of RPAs showing the changes in
lrtA transcript levels when illuminated cyanobacteria
are transferred to the dark. Lane 1, Probe plus RNase; lane 2, probe (1 µL of a 1:50 dilution from the stock used directly for the
experimental samples) without RNase; lane 3, RPA from cells
continuously grown in the light; lanes 4 to 12, RPAs from aliquots of
illuminated cells placed in the dark and taken at 20-min intervals;
lane 13, cells dark adapted for 4 h; lane 14, cells dark adapted
for 6 h; and lane 15, cells dark adapted for 7 h. B, The
quantitation of data from three independent experiments (including the
one shown in A) plotted versus time in the dark. The level of the
transcript present at each time point was quantitated using a
densitometer and calculated as a percentage of a dark control (amount
of transcript present in cells dark adapted for 3 h) for
comparison.
|
|
The Transcript for lrtA Is More Stable in the Dark
Than in the Light
The RPA technique was used to measure the effects of rifampicin on
the accumulation and stability of the lrtA transcript. Rifampicin (50 µg mL1) was added to 3-h
dark-adapted cells that were kept in the dark. Aliquots were taken from
the cells at 0, 2, 5, 10, 15, 20, 25, and 30 min after the addition of
rifampicin. The data showed that lrtA transcript levels in
the dark remained constant for at least 30 min (Fig.
3, A and C), whereas rifampicin added at
the onset of dark adaptation prevented the synthesis of the
lrtA transcript (data not shown). When dark-adapted cells
were exposed to light in the absence of rifampicin, a rapid decrease in
transcript level was seen, with a half-life of approximately 4 min
(Fig. 3, B and C). In the presence of rifampicin, a 4-min half-life
again was observed for the lrtA transcript when dark-adapted
cells were reexposed to light (Fig. 3C). Meanwhile, in dark-adapted
cells, 100% of the message remained 3 h after the introduction of
rifampicin (Fig. 3C).
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| Figure 3.
A, Cells were dark adapted for 3 h followed
by the addition of 50 µg mL1 rifampicin. Aliquots were
taken 0, 2, 5, 10, 15, 20, 25, and 30 min after the addition of
rifampicin (lanes 4-11). Lane 1, RPA from continuously illuminated
cells only; lane 2, probe only (1 µL of a 1:50 dilution); and lane 3, RPA from 3-h dark-adapted cells. B, Cells were allowed to dark adapt
for 3 h, and then were exposed to light in the absence of
rifampicin. Aliquots were taken 0, 5, 10, 15, and 20 min (lanes 4-8)
after the start of illumination. Lane 1, Probe only; lane 2, RPA from
illuminated cells only; and lane 3, RPA from 3-h dark-adapted cells. C,
 , Quantitation of data from three independent experiments (including
the one shown in A) in which samples from dark-adapted cells, incubated
with 50 µg/mL rifampicin, were assayed with RPA for
lrtA transcript levels at the indicated time points in
the dark;  , quantitation of data from B (dark-adapted cells
transferred to light and assayed for transcript levels at the indicated
time points); and  , quantitation from an experiment similar to the
one shown in B, but with the addition of 50 µg/mL rifampicin to the
dark-adapted cells before the onset of illumination.
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|
RNase Activities of Cell Extracts from Dark- or Light-Adapted Cells
Do Not Differ Significantly
Possible explanations for increased transcript stability in the
dark include the following: (a) structured RNA components (stem loops)
within the transcript are formed to protect against degradation; (b)
specific protein(s) bind to the transcript, preventing degradation; and
(c) in dark-adapted cells, RNase activity decreases. To explore the
reasons that the lrtA transcript is more stable in the dark,
differences in RNase activity between dark-adapted and illuminated
cells were measured by the degradation of in vitro-labeled RNA
visualized on acrylamide gels. Several RNAs were used to monitor RNase
activity, including petCA (Brand et al., 1992),
cpcBAC (de Lorimier et al., 1984), ndhB (M. Varughese and W.R. Widger, unpublished data), both sense and antisense
lrtA fragments, and the full-length sense lrtA
message. Figure 4 shows the time course
for RNA digestion by cell extracts obtained from light- and
dark-adapted cells. Some noticeable differences were found between the
degradation rates of in vitro-generated sense lrtA RNA and
those of in vivo-generated lrtA RNA. Extracts from
dark-adapted cells showed increased rates of RNA degradation compared
with extracts from light-adapted cells (Fig. 4A). In vivo, the
lrtA message was not degraded in the dark, whereas the in
vitro-synthesized lrtA message was degraded by cell extracts
from both dark- and light-adapted cells. The degradation of several
other mRNAs, i.e. cpcBAC (Fig. 4B), petCA,
ndhB, and antisense lrtA, by cell extracts from
light- and dark-adapted cells (data not shown) gave
similar results. The amount of protein extract used in each set of
reactions was selected to follow the degradation rates on the gels. The
data from these experiments suggest that only small changes in RNase
activity are seen and that these are the opposite of what would be
expected if changes in RNase activity controlled lrtA
transcript stability; more activity is seen in the dark than in the
light. Thus, changes in general RNase activity do not appear to be the
reason for the differential stability of the lrtA
transcript. However, transcript stability could be caused by protection
against RNase activity by protein binding to stem-loop structures.
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| Figure 4.
Ten-microgram aliquots of cell extracts from
illuminated (lanes 2-4) or dark-adapted (lanes 5-7) cells were
incubated with 100,000 cpm of full-length sense lrtA
mRNA (A) or sense RNA from the 880-bp
XhoI-BamHI fragment from the
cpcBAC gene (B). Each reaction was allowed to take place
for 30, 60, or 90 min in the light (lanes 2, 3, and 4, respectively) or
in the dark (lanes 5, 6, and 7, respectively) for the experiments in
both A and B. Lane 1, RNA only in both experiments.
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|
An Extensive Secondary Structure for lrtA RNA Is
Predicted
A putative stem-loop structure was predicted in the 5 UTR of the
lrtA transcript starting at nucleotide 4 from the determined 5 start site (Fig. 5A). This structure
shares features common to structures known to confer RNA stability to
several prokaryotic transcripts, including ompA in E. coli (Chen et al., 1991), the ermA and ermC
transcripts of Bacillus subtilis and Staphylococcus aureus (Sandler and Weisblum, 1988), and the gene 32 mRNA of phage T4 (Gorski et al., 1985). The structure predicted in the 5
lrtA UTR is more extensive than that seen in
ompA, and appears to be present in Synechocystis
sp. PCC 6803 lrtA 5 UTR (Kaneko et al., 1996) (Fig. 5B).
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| Figure 5.
A, Secondary structure at the 5 UTR of
lrtA from Synechococcus sp. PCC 7002. Transcription starts at position 1. B, Secondary structure at the 5
UTR of lrtA from Synechococcus sp. PCC
6803 (Kaneko et al., 1996, and Cyanobase
[http://www.kazusa.or.jp/cyano/cyano.html]). The secondary structures
were predicted using the RNAfold program (Zuker and Stiegler, 1981)
from the Genetics Computer Group package (Devereux, 1991).
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The Synechococcus sp. PCC 7002 lrtA 5 UTR
secondary structure has a calculated G of 68.4 kcal
mol1 (Zuker and Stiegler, 1981), and contains
three major stem loops. The first stem loop is 124 bases long and
contains an 8-base direct repeat surrounding a 6-base inverted repeat
at positions 57 to 94 (Fig. 1). This stem loop, which starts at the
fourth nucleotide from the transcript start site, could be involved in
RNA stability, especially against degradation initiated at the 5 end.
The second stem loop is 113 bases long and is positioned in the middle
of the 5 UTR (from nucleotides +147 to +260). The third stem loop is
116 bases long, starts at nucleotide 261, and extends to nucleotide 376 (Fig. 5A). A putative ribosome-binding site sequence (AGAGA) 7 bases
before the start of translation is included in this stem structure at
position 368. This loop may be responsible for the observed shortened
transcripts that terminate before the start of translation (Tan et al.,
1994). A fourth stem loop, a typical Rho-independent transcription
terminator, is located between nucleotides 1035 and 1068 downstream
from the coding region (data not shown) with a G of
9.6 kcal mol1 (Zuker and Stiegler, 1981). The
predicted secondary structure in the 5 UTR of the lrtA
transcript from Synechocystis sp. PCC 6803 is also extensive
and has a calculated G of 85.4 kcal
mol1 (Fig. 5B) (Zuker and Stiegler, 1981).
There is little sequence similarity between the 5 UTRs of
Synechococcus sp. PCC 7002 and Synechocystis sp.
PCC 6803; however, it is of interest that both have extensive predicted
secondary structures, although they are not identical.
Protein Synthesis Is Required for lrtA
Repression in the Light
The effects of CM on the transcription of lrtA in the
light and upon transition from dark to light were studied to determine if protein synthesis is required for the repression of lrtA
(Fig. 6A). Aliquots were taken from
continuously illuminated cells at 10, 30, 60, and 120 min after the
addition of 25 µg mL1 CM and subjected to RPA
analysis. Illuminated cells in the absence of CM showed no detectable
levels of the lrtA transcript (Fig. 6A, lane 2), whereas
transcript levels from the same cells, only dark adapted, were easily
detected (Fig. 6A, lane 3). Addition of CM to light-adapted cells
followed by further incubation in the light allowed the synthesis of
detectable levels of the lrtA transcript, albeit low in
concentration (Fig. 6A, lanes 4-7, and B). The maximum amount of the
lrtA transcript was seen 30 min after the addition of CM.
Furthermore, the message was detectable for up to 2 h in the light
after the addition of CM. These data were quantitated and plotted as a
function of time (Fig. 6B).
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| Figure 6.
Effects of CM on lrtA
transcription. A, Cells were placed in the light for 3 h and
aliquots were analyzed for transcript levels at various times after the
addition of 25 µg mL1 CM. Lane 1, Probe only; lane 2, the RPA from 3-h-illuminated cells; lane 3, the RPA from cells dark
adapted for 3 h; and lanes 4 to 7, 10, 30, 60, and 120 min after
the addition of CM, respectively. B, Quantitation of the data shown in
A plotted versus time as a percentage of the corresponding dark control
(cells dark adapted for 3 h, lane 3 in A) (). For a comparison,
the absence of the lrtA transcript in constant light is
shown (). C, Cells were dark adapted for 3 h, CM was added, and
the cells were illuminated. Samples were taken at 10, 30, 60, and 120 min after illumination (lanes 1, 2, 3, and 4, respectively). The same
probe and controls as in A were used for this experiment. D, Cells dark
adapted for 3 h were incubated with 25 µg mL1 CM
and 50 µg mL1 rifampicin for 10 min. The cells were
then exposed to light. Lane 1, Probe only; lane 2, probe plus RNase;
lane 3, RPA from cells grown in constant light; lane 4, RPA from 3-h
dark-adapted cells; lanes 5, 6, 7, and 8, samples taken 10, 30, 60, and
120 min after the start of illumination, respectively, and subjected to
RPA analysis. E, Quantitation of the data shown in C () and in D
(). Some data from Figure 3B (transcript levels in cells dark
adapted for 3 h and then illuminated) are shown for comparison (). The arrows in A, B, and C indicate the positions of the
protected fragment of the lrtA message.
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|
Addition of 25 µg mL1 CM to dark-adapted
cells, followed by illumination, allowed the detection of
lrtA transcript for 2 h after illumination (Fig. 6C).
This is in contrast to the 4-min half-life observed in the light
without the addition of CM (Fig. 3, B and C). To determine if the
CM-induced increase in transcript half-life is caused by suppression of
a particular RNase or whether CM relieves the repression of
transcription in the light, this experiment was repeated in the
presence of both CM and rifampicin (Fig. 6D). A short half-life of 10 min was observed, suggesting that the prolonged detection of the
lrtA message in the presence of CM was caused by the relief
of transcription repression in the light. Quantitative data from the
experiments shown in Figure 6, C and D, and Figure 3B are plotted
together for a comparison (Fig. 6E).
| |
DISCUSSION |
In the present study, lrtA transcript levels were
measured by RPAs in the light and in the dark. From these measurements, we have repeated and extended the initial conclusions that the lrtA transcript is not detected in the light, but is
synthesized to significant levels when cells are placed in the dark
(Fig. 2, A and B) (Tan et al., 1994). The lrtA transcript
was shown to have an unusual stability in the dark compared with
illuminated cells. Cells treated with rifampicin showed that the
lrtA transcript was stable (without detectable losses for 30 min) after an initial 3-h dark incubation (Fig. 3, A and C), and nearly
100% of the message was detected 3 h after treatment (Fig. 3C). A
half-life of greater than 3 h in the dark was estimated from these
data. The half-life of the lrtA transcript in illuminated
cells in the presence or absence of rifampicin was 4 min (Fig. 3, B and
C), suggesting that the synthesis of new transcripts is not required for the rapid degradation of lrtA mRNA after illumination.
The overall RNase activities found in cell extracts from light-grown
and 3-h dark-adapted cells were similar (Fig. 4, A and B). However,
extracts from dark-adapted cells consistently gave slightly higher
rates of RNA degradation than extracts from illuminated cells for all
RNA species assayed. Sense RNA from ndhB, petCA (data not shown), lrtA, and cpcBAC (Fig. 4, A and
B) showed nearly identical rates of degradation by extracts from
dark-adapted cells. This suggests that in vitro-synthesized RNA is
degraded differently from in vivo-generated RNA. The sense strand of
lrtA was made in vitro using the E. coli
polymerase (Burgess et al., 1975), and this message was degraded
equally by extracts isolated from dark- or light-adapted cells. These
data suggest that the increased synthesis of general RNases in the
light did not cause increased lrtA transcript degradation;
however, rates of in vivo RNase activity may not accurately reflect the
activity measured in cellular extracts. RNase activity could be altered
by isolation, causing an activation of activity in the dark or a
deactivation of it in the light. Folding patterns of in vivo message
compared with in vitro-synthesized message could be different, leading
to digestion of the in vitro message in dark-adapted cell extracts, and
ratios of RNA to RNase could also vary. These concerns are separate
from the fact that measurable RNase activity of extracts does not vary
substantially from light to dark.
A lag time of 20 to 30 min was seen for the appearance of the
lrtA message when illuminated cells were placed in the dark (Fig. 2, A and B), suggesting that factors other than darkness are
required for lrtA transcription. Repression of the
transcript in the light requires protein synthesis, since the addition
of CM to illuminated cells eases the transcription repression of lrtA (Fig. 6A). The data suggest that a specific protein is
directly involved in lrtA light repression and that blockage
of the synthesis of this repressor protein by CM allows lrtA
to be transcribed.
Under autotrophic growth conditions, most other transcripts in
Synechococcus sp. PCC 7002 are down-regulated in the dark, whereas lrtA behaves in the opposite manner. This behavior
suggests a regulatory role for lrtA, yet no function for the
gene product has been identified. Unfortunately, a specific phenotype
has not been identified with the loss of the lrtA gene by
mutation. Knockout mutants of lrtA grown in continuous light
appear to be the same as wild type, showing no differences in growth or
photosynthetic activity (Tan et al., 1994). This is not surprising
because the lrtA gene product is not synthesized in the
light.
When CM-treated, dark-adapted cells were placed in the light, an
increase in the half-life of the transcript was observed (Fig. 6C).
Significant amounts of transcript were present 1 h after
illumination in the presence of CM, whereas no message was seen in
1-h-illuminated cells without CM (Fig. 3B). These data are in agreement
with the results from the experiment in which CM was added to
continuously illuminated cells (Fig. 6A), suggesting that CM inhibited
repression of lrtA transcription. However, the apparent
increased half-life in illuminated cells treated with CM could be the
result of two possible mechanisms: (a) the inhibition of the synthesis
of a specific RNase, or (b) the loss of transcript repression by the
action of CM. To address the two possibilities, the RPA experiment was
performed in the presence of both rifampicin and CM. The half-life of
the transcript in the light approached 10 min, returning to times seen
for untreated cells (Fig. 6D). This suggests that RNases are already
present in the dark and are active, as previously intimated from the
RNase activity data. No new RNase should be synthesized because most
transcription and translation are not functioning. A working model
suggests that the loss of a repressor protein, because of CM
inhibition, leads to the production of the lrtA message.
Normally, production of this light-activated (translated) repressor
protein would be responsible for transcript repression in the light.
The loss of this putative protein in the dark (down-regulation of
transcription/translation) or in the light because of the effects of CM
would allow the transcription of lrtA to proceed, which is
consistent with the observed results.
Two factors govern transcript abundance: the rate of transcript
formation and the rate of transcript degradation. Evidence for a
repressor protein controlling transcript formation has been presented
above; however, factors controlling transcript stability are unknown.
The lifetimes of individual messages can vary widely within a single
cell and are often regulated in response to changes in the cell's
environment or growth phase. Each rate is a separate function
controlled by different factors. Differences in the transcript stability of IrtA under varying conditions could be
explained by (a) a specific light- induced RNase activity that
selectively degrades the lrtA transcript in the light but
not in the dark; (b) protein(s) binding to the lrtA
transcript in the dark, leading to protection against RNase attack; or
(c) changes in RNA stability (structures) by altered stem-loop
conformation induced by light/dark changes in protein binding.
There appear to be no major differences in the RNase activity between
cell extracts isolated from dark- and light-adapted cells that could
account for the observed differential lrtA transcript stability (Fig. 4). We make this statement with the caveat that RNase
activity from cell extracts may be different from that in intact cells.
In addition, earlier studies in our laboratory (Brand et al., 1992)
have shown that many photosynthetic transcripts, such as
petBD, petCA, and cpcBAC, are
transcribed only in the light and that their transcript levels are
undetectable soon after the cells are placed in the dark. This
observation suggests that these transcripts are not stable in the dark.
However, the Synechocystis sp. PCC 6803 psbA
transcript is very stable in the dark, with a half-life of 7 h,
but it is unstable in the light. This behavior is controlled by
photosynthetic electron transport (Mohamed and Jansson, 1991). However,
the Synechocystis sp. PCC 6803 psbA transcript is
transcribed only in the light and not in the dark, the opposite of the
behavior of the lrtA transcript. In the same organism, the
rbcLS transcript is more stable in the light than in the
dark (Mohamed and Jansson, 1991). The Synechococcus sp. PCC
7942 psbA gene family is posttranscriptionally regulated by
light (Golden, 1995). The psbAI and psbAIII
transcripts are degraded faster under high-light conditions, and this
regulation requires de novo transcription and translation after
exposure to high light. In contrast to photosynthetic genes,
transcripts of the rrn genes are present in both dark- and
light-adapted cyanobacteria cultures (Mohamed and Jansson, 1989; Tan et
al., 1994).
The lrtA 5 UTR is 377 bases long and exhibits extensive
secondary structure from base 4 to 397, with an estimated
G of 68.4 kcal mol1 (Zuker
and Stiegler, 1981) (Fig. 5A). The role of this putative stem-loop
region is unclear. Extensive secondary structure is also predicted for
the 5 UTR of the lrtA from another cyanobacterium, Synechocystis sp. PCC 6803. The 5 UTR of the
lrtA from Synechocystis sp. PCC 6803 is 477 bp
long. Comparisons of secondary structure predictions for each species
show similarity even though the sequences are not conserved in the 5
UTR of the lrtA transcript from both organisms. Although the
individual stem-loop structures are different and the significance of
these RNA structures is unclear, the extensive secondary structure
present in the 5 UTR of lrtA from both
Synechococcus sp. PCC 7002 and Synechocystis sp.
PCC 6803 suggests a similar function.
Similar RNA structures stabilize transcripts for ompA
(Belasco et al., 1986; Bechhofer, 1993), papA,
pufBA, and T4 phage gene 32 (Emory et al., 1992; Bechhofer,
1993). In E. coli, for example, mRNA half-lives range from a
few seconds to 1 h, with an average lifetime of 2 to 4 min (Emory
and Belasco, 1990). Structured elements at the 3 and 5 ends of phage
and bacterial messages have been shown to influence mRNA stability. RNA
stem-loop structures in the 5 UTR function as 5 stabilizers and have
been identified in the ompA transcript in E. coli
(Chen et al., 1991), the ermA and ermC
transcripts of B. subtilis and S. aureus (Sandler
and Weisblum, 1988), and the gene 32 mRNA of phage T4 (Gorski et al., 1985). 5 cis-acting elements are important determinants of
the lifetimes of Chlamydomonas reinhardtii gene
transcripts (Salvador et al., 1993). Stem-loop structures at the 3 end
of a prokaryotic transcript can increase its stability by blocking the
processing action of 3 to 5 exonucleases (for review, see Higgins et
al., 1993). In plastids, 3 inverted repeats, which can potentially form stem-loop structures, act as mRNA-processing and -stabilizing elements, but they do not terminate transcription (Stern and Gruissem, 1987).
Stabilizing an RNA structure by protein binding in the dark is an
attractive hypothesis with some precedent. Chloroplast-encoded proteins, specifically the psbA message in C. reinhardtii, are expressed in the light, whereas messages are
present both in the light and in the dark (Mullet, 1988; Danon and
Mayfield, 1994). The expression of psbA in chloroplasts is
thought to be controlled by an NADPH-dependent thioredoxin reduction of
specific protein thiols, allowing this protein to bind near the 5
untranslated end of the message. This binding mediates the onset of
translation in the light (Danon and Mayfield, 1994; Mayfield et al.,
1995). In Synechococcus sp. PCC 7002, lrtA is not
translated in the dark to significant levels but it is translated at
the onset of illumination (Tan et al., 1994). Mechanisms similar to
those regulating chloroplast psbA expression may be at work,
conferring transcript stability and translation regulation of the
lrtA gene.
The data presented here suggest that the pathway leading to the
repression of lrtA requires protein synthesis. The protein responsible for repression has not been identified, but the loss of
repression by CM suggests its presence. Two important questions remain
unanswered: What factors are responsible for (a) lrtA
transcription in the dark while most other mRNA species are not
synthesized, and (b) the increased stability of the lrtA
transcript in the dark?
| |
FOOTNOTES |
1
This research was supported by the National
Institutes of Health (grant no. GM46297), the Robert A. Welch
Foundation (grant no. E-1381), and the National Science Foundation
(equipment grant no. BIR 9109294).
*
Corresponding author; e-mail billw{at}photo.bchs.uh.edu; fax
1-713-743-8351.
Received September 29, 1997;
accepted February 11, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CM, chloramphenicol.
G, free
energy of folding.
RPA, RNase protection assay.
UTR, untranslated
region.
| |
ACKNOWLEDGMENTS |
The authors thank Dr. Costas Koumenis and Dr. Arnold Eskin for
their help on the use of the RPAs and Dr. Daniel Davison for helpful
discussions and review of the manuscript.
| |
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Abstract
Introduction