(Received for publication, July 25, 1995; and in revised form, October 19, 1995)
From the
We have examined the effects of illumination, carbon source, and
levels of chloroplast protein synthesis on trans-acting proteins that
bind to the leaders of five representative chloroplast mRNAs. The
accumulation of these five chloroplast mRNAs and the proteins they
encode were measured in cells grown under identical conditions.
Extracts from all cell types examined contain a minimum set of six
chloroplast 5`-untranslated region (UTR)-binding proteins (81, 62, 56,
47, 38, and 15 kDa). Fractionation results suggest that multiple forms
of the 81-, 62-, and 47-kDa proteins may exist. A 36-kDa protein was
found in all cells except those deficient in chloroplast protein
synthesis. Binding of the 81-, 47-, and 38-kDa proteins to the rps12 leader is effectively competed by the atpB or rbcL 5`-UTRs, indicating that the same proteins bind to all
three leaders. In contrast, these three proteins do not bind to the
nuclear-encoded -1 tubulin leader, which bound novel
proteins of 110, 70, and 43 kDa. Cis-acting sequences within the
5`-UTRs of two chloroplast mRNAs (rps7 and atpB) have
been identified which are protected from digestion by RNase T1 by
extracts enriched for the 81-, 47-, and 38-kDa proteins.
Regulation of chloroplast mRNA translation represents an
important determinant of plastid gene
expression(1, 2) . Abundant genetic evidence exists in
the green alga Chlamydomonas reinhardtii that nuclear-encoded
factors are required for the stability, processing, and translation of
chloroplast-encoded mRNAs (for reviews, see (1, 2, 3, 4) ). In this alga, the
light-regulated translation of the psbA mRNA encoding the D1
protein of photosystem II correlates with the binding of a 47-kDa
protein(s) to a 30-nucleotide sequence within its 5`-untranslated
region (UTR) ()starting at position -60 5` to the AUG
codon. This sequence ends immediately adjacent to the putative
Shine-Dalgarno (SD) sequence which begins at position -30
nucleotide(5) . Binding of the 47-kDa protein(s) to the 5`-UTR
is thought to lead to the formation of an active translation complex
which is inactivated in the dark by phosphorylation of a 60-kDa member
of this complex that has only minor RNA contact(6) .
Additionally, binding of these trans-acting proteins to psbA mRNA is postulated to respond to the redox potential in the
chloroplast established by photosynthesis(7) . In contrast,
binding of a 46-kDa protein to the psbC 5`-UTR in the
recessive nuclear mutant F64 has been correlated with the failure to
translate psbC mRNA encoding P6, the 43-kDa chlorophyll a-binding core subunit of photosystem II(8) .
In addition to light-mediated translational regulation, as seen in the case of psbA mRNA, translation of plant and algal chloroplast mRNAs may respond to other environmental and physiological regulatory signals. When chloroplast protein synthesis is reduced, C. reinhardtii preferentially translates mRNAs for chloroplast-encoded ribosomal proteins (r-proteins), while translation of mRNAs for photosynthetic proteins is severely diminished(9) . Furthermore mRNAs for chloroplast-encoded r-proteins appear to be constitutively transcribed, accumulate early during chloroplast development in land plants and are preferentially loaded on polysomes(10, 11) . Nutrients, especially carbon source, can act as central regulatory signals controlling physiology, metabolism, cell cycle, and development in land plants and green algae (for reviews, see (12) and (13) ). In maize cells in culture, acetate represses transcription of seven nuclear-encoded photosynthetic genes (14) . Similarly, in Chlamydomonas and other ``acetate flagellates'' this reduced carbon source has been shown to repress the expression of the nuclear-encoded rbcS and cab genes(15, 16) . Much less is known about the effect of carbon source on chloroplast gene expression.
In this study we have attempted to broaden our understanding of the participants required for translational regulation in chloroplasts of C. reinhardtii by examining the spectrum of proteins capable of binding to the 5`-UTRs of representative chloroplast-encoded mRNAs specifying photosynthetic and r-proteins(1, 2, 3, 4) .
RNA leaders
were synthesized in 20-µl reactions containing 1 µg of
linearized DNA template in 40 mM Tris-HCl (pH 7.5), 6 mM MgCl, 2 mM Spermidine (Sigma), 10 mM dithiothreitol, 20 units of RNasin (Promega), 50 µCi of
[
-
P]UTP (800 mCi/mmol, DuPont NEN), 12
µM nonradiolabeled UTP, 0.3 mM each of ATP, CTP,
and GTP and 20 units of T7 RNA polymerase (U. S. Biochemical
Corp./Amersham Corp.) for 1 h at 37 °C. 1 unit of RNase-free DNase
I (Sigma) was added, and the reaction was incubated for an additional
10 min at 37 °C. Under these conditions RNAs were labeled to
specific activities of 5
10
to 2
10
cpm/µg. Unlabeled transcripts used in competition experiments
were synthesized as above except that all four unlabeled
ribonucleotides were included, and the reactions were scaled up to a
100-µl final volume. The reactions were
phenol-chloroform-extracted, and the RNA probes were separated from
unincorporated nucleotides on Sephadex G-25 spun columns (23) .
All 5`-UTR probes were derived from the clones described above. Total
RNA was isolated as described above from E. coli cells (strain
XL1-Blue) grown in LB medium to mid log phase (0.65 A
) and from C. reinhardtii mutant
strains CC-373 and CC-744 grown mixotrophically to mid log phase
(1-3
10
cells/ml). These RNAs were used as
alternative competitors to E. coli tRNA (Calbiochem) in
certain UV cross-linking experiments.
For the gel mobility shift
assay, pooled or individual heparin-Actigel column fractions (7 µg)
were preincubated for 10 min at room temperature with 5 units of RNasin
in the presence of 3 mM MgCl in a total volume of
5 µl and then added to 20 µg of E. coli competitor
tRNA and
P-labeled rps12 leader (15 pM)
in a final volume of 15 µl. After 15-min incubation at room
temperature, 2 µl of xylene cyanol were added, and the mixtures
were loaded onto a 15 cm
15-cm 5% (49:1
acrylamide:bisacrylamide) native polyacrylamide gel containing 1
TBE and electrophoresed in TBE buffer at 25 mA for 2 h until
the dye marker was about 2.5 cm from the bottom of the gel. The gel was
then fixed in 10% methanol, acetic acid, dried, and exposed to x-ray
film (Kodak XAR5) at -70°.
The conditions for UV
cross-linking were described previously(5, 33) .
Binding reactions (15 µl) were performed as follows: 7 µg of
protein from individual heparin-Actigel column fractions was
preincubated in the presence of 3 mM MgCl and 0.5
units of RNasin in a volume of 5 µl for 10 min at 22 °C. E.
coli tRNA (0.1 µg) as a nonspecific competitor and
[
P]UTP-labeled chloroplast 5`-UTR probe (about
15 pM) were added to give a final volume of 15 µl. After
15 min at 22 °C, the binding reactions were placed on ice and
cross-linked with 254-nm UV irradiation of 1.0 J/cm
using a
Stratalinker (Stratagene). The RNA transcripts were digested with 10
µg of RNase A (Sigma) for 30 min at 55 °C, boiled for 1 min in
protein loading buffer, separated on 15 cm
15-cm, 7.5-17%
SDS-polyacrylamide gels, dried, and exposed to x-ray film (Kodak XAR5
or Fuji RX) at -70° using intensifying screens (DuPont
Chronex). In competition experiments, E. coli tRNA or rRNA
(100-300-fold molar excess) or total RNA (50-100-fold mass
excess) from the C. reinhardtii strains CC-373 and CC-744 were
preincubated with protein extracts prior to the addition of
[
P]UTP-labeled leaders. Unlabeled 5`-UTRs from
the chloroplast atpB and rbcL genes or the polylinker
sequence from the pBluescript KS
plasmid (0-2.5
nM) were also included in certain binding reactions as
specific competitors prior to addition of the labeled 5`-UTR RNA.
Analysis of UV cross-linking reactions for each column fraction on the
autoradiograms allowed us to define binding proteins of the same
molecular weight which differ in their elution profile from the
heparin-Actigel column due either to protein modifications or to
differences in their amino acid sequence.
Fig. 1compares the steady state levels of mRNA and protein
for the psbA, rbcL, atpB, rps7, and rps12 genes found in cells grown under different conditions of
1) illumination (mixotrophic = light + acetate versus heterotrophic = dark + acetate), 2) carbon source
(mixotrophic = light + acetate versus phototrophic
= light + CO), and 3) levels of chloroplast
protein synthesis under mixotrophic growth (ac-20 cr-1 versus wild type; spr-u-1-27-3, + versus -
spec). Representative Northern blots and immunoblots for each of the
five chloroplast genes are shown in Fig. 1A and the
values of replicate experiments are quantified in Fig. 1B as a percentage of the values for phototrophically grown wild type
cells with the range of variation indicated. To verify equivalent
protein loading for each extract, the accumulation of the
nuclear-encoded
-tubulin protein is shown (Fig. 1A).
Figure 1:
Effects of illumination, carbon source
and reduced chloroplast protein synthesis on the accumulation of the psbA, rbcL, atpB, rps7, and rps12 mRNAs and the
corresponding D1, LSU, , S7, and S12 proteins. A, The top panel for each of the five chloroplast genes represents a
Northern blot showing accumulation of mRNA and the bottom panel shows an immunoblot of the protein encoded by these genes from
mixotrophically (M), heterotrophically (H), and
phototrophically (P) grown wild type and the mixotrophically
grown mutants ac-20 cr-1 and spr-u-1-27-3 (spr-u). The ac-20 cr-1 double mutant is
permanently deficient in chloroplast protein synthesis while the spr-u-1-27-3 mutant is deficient in chloroplast protein
synthesis when grown in the presence of spectinomycin
(+spec), but not in the absence of antibiotic
(-spec). An immunoblot of
-tubulin is presented to
verify equivalent protein loadings. B, quantification of the
mean accumulation of each mRNA (open bar) and protein (solid bar) determined from two separate cultures, normalized
to the values for phototrophically grown wild type cells. The range of
values obtained under each condition is denoted by bars.
Unlike angiosperms and algae such as Euglena, which do not maintain a differentiated chloroplast in
the dark, heterotrophically grown wild type cells of C. reinhardtii synthesize chlorophyll and contain well developed thylakoids
stacked into grana (18, 41) . Accumulation of LSU,
, S7, and S12 proteins in C. reinhardtii was found to be
essentially unaffected by dark versus light growth in the
presence of acetate. In contrast, heterotrophically grown cells appear
to accumulate only 24% of D1 found in mixotrophically grown cells and
40% of that in phototrophically grown cells. While levels of psbA and rps7 mRNA show mostly minor variations between these
growth conditions, accumulation of the rps12, rbcL,
and atpB mRNAs in mixotrophically grown cells is 30-50%
of that in heterotrophically or phototrophically grown cells. Thus
levels of protein accumulation are not directly coupled to steady state
levels of mRNA in the chloroplast of this alga.
Growth of C.
reinhardtii in the light on acetate has been reported to reduce
the abundance of the nuclear-encoded cabII-1 transcript,
encoding a chlorophyll a/b-binding protein and to modify the
ratio of the two nuclear transcripts encoding the small subunit (SSU)
of Rubisco compared to phototrophically grown
cells(16, 42) . We found that acetate also
differentially altered the expression in light grown cells of the D1
and LSU photosynthetic proteins encoded by the chloroplast rbcL and psbA genes. Mixotrophically grown cells accumulated
about 2-fold higher levels of D1 protein and 1.5-fold less LSU protein
compared to phototrophically grown cells. No appreciable changes were
observed in accumulation of the S7, S12, or proteins under these
conditions. Accumulation of mRNAs encoding the photosynthetic proteins
LSU and
were reduced 2.2-5-fold in mixotrophically versus phototrophically grown cells (Fig. 1). In
contrast, accumulation of the psbA mRNA encoding the D1
protein was only slightly reduced by the presence of acetate in the
medium. Again little or no correlation was seen between accumulation of
a specific mRNA and its cognate protein.
We also evaluated the
effects of reduced chloroplast protein synthesis on the accumulation of
chloroplast-encoded photosynthetic and r-proteins and their mRNAs. Two
mutant strains deficient in chloroplast protein synthesis were
utilized: 1) the nuclear ac-20 cr-1 double mutant which
results in a permanent deficiency of chloroplast ribosome
monomers(18) , and 2) the chloroplast 16 S rDNA mutant, spr-u-1-27-3, which when grown mixotrophically in the presence
of spectinomycin, accumulates nearly wild type levels of chloroplast
ribosomes, but is deficient in chloroplast protein
synthesis(9) . Mixotrophically grown cells of ac-20 cr-1 were found to be severely deficient in the chloroplast encoded D1,
LSU, and photosynthetic proteins, but accumulated nearly wild
type levels of chloroplast encoded r-proteins S7 and S12 (Fig. 1). Levels of rps7, rps12, and rbcL mRNAs detected in ac-20 cr-1 (Fig. 1) were equal
or greater than those in wild type cells grown on acetate in the light,
whereas the psbA and atpB messages were at least 60%
of the values for mixotrophically grown wild type cells.
The
chloroplast mutant spr-u-1-27-3 grown on spectinomycin
accumulated no D1 or LSU and greatly reduced , but nearly normal
levels of r-proteins S7 and S12 (Fig. 1). Accumulation of atpB and rbcL mRNAs under these conditions was
greater than in wild type or in the mutant grown in the absence of
spectinomycin, whereas psbA mRNA was greatly reduced. In
contrast levels of rps7 mRNA were equal to those in cells with
normal chloroplast protein synthesis although the rps12 mRNA
exhibited a reduction. Our results with both mutants are consistent
with the hypothesis of class-specific translational regulation of
ribosomal versus photosynthetic proteins under conditions of
reduced chloroplast protein synthesis(1, 9) .
Figure 2:
A, autoradiogram of a gel retardation
assay demonstrating the presence of proteins in heparin-Actigel column
fractions from an S-200 extract of C. reinhardtii that bind to
the 5`-UTR of the chloroplast rps12 mRNA. P-Labeled transcripts for the rps12 5`-UTR were
incubated either alone (control), in the presence of pooled
column fractions heated to 100 °C for 10 min (boiled
extract), or in the presence of individual column fractions (heparin-Actigel column fractions). The S-200 extract was
prepared from the spr-u-1-27-3 mutant grown in the absence of
spectinomycin. All reactions contained a
100-fold mass excess E. coli tRNA to compete for any nonspecific RNA binding
proteins present in the column fractions. Each lane represents a gel
retardation assay with proteins from a single fraction eluted with an
increasing potassium acetate (KOAc) gradient. B,
concentration dependent binding of heparin-Actigel-purified proteins to
the rps12 5`-UTR. Filter binding assay was conducted by
incubating uniformly radiolabeled rps12 5`-UTR (14
pM) in the presence of increasing concentrations (1
10
to 1
10
µg/µl
protein) of pooled heparin-Actigel column fractions from the above
extracts.
A high degree of reproducibility was found in the pattern of 5`-UTR binding proteins between individual UV cross-linking experiments done with the same extract and sequential preparations of in vitro transcribed leaders of the same chloroplast gene. Duplicate heparin-Actigel fractions prepared from separate cultures of the same genotype (spr-u-1-27-3, +spec) or from phenotypically similar genotypes (spr-u-1-27-3, -spec and wild type) also contained the same sets of 5`-UTR binding proteins. Since the chloroplast occupies a large percentage of cell volume in C. reinhardtii(17) , chloroplast proteins would be expected to comprise a large proportion of the total protein in cell extracts and hence predominate among total proteins in the S-200 preparations.
The presence of 100-fold molar excess E. coli tRNA over
labeled 5`-UTR in the binding reactions minimized binding of
nonspecific proteins from the pooled heparin-Actigel columns to the
chloroplast 5`-UTRs. Increasing the tRNA concentration or adding total E. coli RNA to 250-fold molar excess compared to labeled probe
had no detectable effect on the binding of the 81-, 47-, and 38-kDa
proteins to the psbA (Fig. 3) or atpB (data
not shown) chloroplast leaders, whereas addition of 300-fold molar
excess tRNA or total E. coli RNA resulted in a slight
reduction in the binding of these proteins to both leaders. None of the
proteins bound to the 5`-UTR from the nuclear -1 tubulin
gene of C. reinhardtii (Fig. 3). Instead, the
-1 tubulin leader bound three new proteins of 110, 70,
and 43 kDa which were also not competed off by excess tRNA or total RNA
from E. coli. Thus we believe the purification of whole cell
lysates on heparin-Actigel columns and UV cross-linking is a valid
approach for isolating and characterizing proteins that bind
specifically to 5`-UTRs of chloroplast mRNAs.
Figure 3:
Autoradiograms of SDS gels showing the
effects of competitor E. coli tRNA and rRNA on the binding of
proteins to the 5`-UTRs of the chloroplast psbA and nuclear -1 tubulin mRNAs from C. reinhardtii.
Subsaturating amounts of pooled heparin-Actigel column fractions (7
µg/µl) from phototrophically grown wild type cells
corresponding to those represented in Fig. 4were UV
cross-linked to [
-
P]UTP-labeled psbA and
-1 tubulin 5`-UTRs (15 pM). E. coli tRNA was added as competitor prior to addition of labeled probe at
concentrations of 100-, 250-, and 300-fold molar excess over the
-
P-labeled 5`-UTR RNA (lanes 1-3).
Reactions in lanes 4 and 5 contained a 150- and
200-fold excess of E. coli rRNA, respectively. Apparent
molecular masses of the bands in kilodaltons were estimated compared to
prestained molecular mass standards. The uppermost band in the
-1 tubulin panel (*) may represent either a high
molecular mass
-1 tubulin-specific protein, or one or more
-1
tubulin-specific proteins that failed to enter the resolving
gel.
Figure 4:
Autoradiograms of SDS gels showing
5`-UTR-binding proteins present in extracts of wild type cells grown
heterotrophically, mixotrophically, or phototrophically that bind to
leaders of representative chloroplast mRNAs. Proteins in
heparin-Actigel column fractions from cell extracts were UV
cross-linked to P-labeled RNAs (synthesized in
vitro) corresponding to the psbA, rbcL, atpB, rps7, and rps12 5`-UTRs. Each lane
within a panel represents a binding reaction with proteins from a
single fraction eluted with an increasing potassium acetate (KOAc) salt gradient. Each UV cross-linking reaction was
repeated at least two times. Apparent molecular masses are indicated in
kilodaltons compared to prestained molecular mass
standards.
Individual lanes in
the autoradiographs of UV cross-linking gels ( Fig. 4and Fig. 5) represent binding reactions with sequentially eluted
column fractions from extracts of cells grown under the conditions
specified. Cross-linking of a given protein to any one of the five
leaders tested is evidence that the protein is present in the
particular extract and fraction analyzed. Absence of binding of a
protein to a specific leader may result from either protein:protein
interactions or structural features within a given leader which may
inhibit binding. Variability in signal intensities of UV cross-linked
proteins between leaders for a given extract arises in part because of
differences in specific activity of the leader probes, differences in
base composition (i.e. number of [P]UTP
residues) of the individual leaders within the protein binding domain
and/or differences in exposure times during autoradiography. The gels
shown in Fig. 4and Fig. 5were exposed in an attempt to
normalize the intensity of the 81-kDa protein, and exposures of varying
lengths were used to verify the results presented.
Figure 5:
Autoradiograms of SDS-gels comparing
5`-UTR-binding proteins present in cells under conditions of normal and
reduced chloroplast protein synthesis. Heparin-Actigel column fractions
from extracts of wild type, the nuclear double mutant ac-20
cr-1, and the chloroplast mutant spr-u-1-27-3 grown
without (-spec) and with (+spec) 40
µg/ml spectinomycin were UV cross-linked to P-labeled
RNAs (synthesized in vitro) corresponding to the psbA, rbcL, atpB, rps7, and rps12 5`-UTRs. Each lane within a panel is a binding reaction
with proteins from a single fraction eluted with an increasing
potassium acetate (KOAc) salt gradient. Each UV cross-linking
reaction was repeated at least two times. Apparent molecular masses are
indicated in kilodaltons compared to prestained molecular mass
standards.
In light grown cells the 47-kDa protein binding to the atpB leader is observed predominantly in two fractions. Absence of a strong 47-kDa protein band from the low salt eluting fraction of the light grown cells is especially evident. A 36-kDa protein that bound to the rps7 and rps12 leaders was much more prominent in extracts from light than dark grown cells. In extracts of heterotrophically and mixotrophically grown cells, the psbA leader cross-links six of the seven proteins, whereas the rps7 leader binds only three or four (Fig. 4). Leaders of the rbcL, atpB, and rps12 mRNAs bind an intermediate number of proteins. Qualitative differences in the binding of the 47- and 15-kDa proteins to specific leaders in individual column fractions are also evident.
Reducing chloroplast protein synthesis over seven- to eight-cell generations by mixotrophic growth of the spr-u-1-27-3 mutant in spectinomycin also affects the spectrum of proteins which binds to the five chloroplast leaders. As in the case of ac-20 cr-1, six of the seven UTR-binding proteins seen in mixotrophically grown wild type cells are present in the S-200 extract from the spr-u-1-27-3 (+spec) cells and all seven are seen in the spr-u-1-27-3 (-spec) cells (Fig. 5). Binding of the 36-kDa protein to all five chloroplast leaders is greatly diminished or absent in extracts from the +spec cells. The reduction in chloroplast protein synthesis in spr-u-1-27-3 does not appear to increase the intensity of the 62-kDa band relative to the 81- and 47-kDa bands as seen in ac-20 cr-1. Binding of the 56-kDa protein to the rps7 and rps12 leaders may be selectively reduced in the +spec extracts. Unique proteins of 60, 45, and 29 kDa UV cross-link specifically with the atpB leader in a single low salt fraction of the +spec extract (data not shown). A new 109-kDa protein also cross-links in the spr-u-1-27-3 (+spec) extract to the atpB leader (Fig. 4) in addition to a unique form of the 15-kDa protein eluting at high salt in both + and -spec extracts of this mutant (data not shown). The significance of the four novel bands specific for the atpB leader present in extracts from spr-u-1-27-3 grown under conditions of reduced chloroplast protein synthesis is unknown. Qualitative differences in the UV cross-linking patters of extracts from ac-20 cr-1 and spr-u-1-27-3 (+spec) may reflect the somewhat ``leaky'' nature of chloroplast protein synthesis phenotype in spr-u-1-27-3 under the latter condition compared to the more stringent phenotype of ac-20 cr-1 with its large reduction in chloroplast ribosomes. Reduction in the amounts of chloroplast synthesized photosynthetic proteins accumulated in spr-u-1-27-3 (+spec) is dependent upon the concentration of spectinomycin used as well as the number of generations the cells are grown in the presence of the antibiotic.
Figure 6:
Competition experiments with unlabeled atpB or rbcL 5`-UTRs. Two heparin-Actigel column
fractions (7 µg/µl) from spr-u-1-27-3 (-spec)
were pooled and preincubated with increasing concentrations of
unlabeled atpB (A) or rbcL (B)
competitor 5`-UTR RNA prior to addition of
[-
P]UTP-labeled rps12 RNA (15
pM). The samples were UV cross-linked and treated with RNase
A, and the proteins were resolved on SDS-polyacrylamide gels.
Radioactivity associated with each individual protein was quantified on
a Molecular Dynamics PhosphorImager and plotted as a function of
competitor concentration (C and D). Apparent
molecular masses are indicated (kilodaltons) compared to prestained
molecular mass standards.
Competition experiments utilizing total RNA isolated from
mixotrophically grown cells of atpB (ac-u-c-2-21 (atpB)) and psbA (ac-u-
(
psbA)) deletion mutants which lack mRNAs for these
two chloroplast genes were also performed. Pooled column fractions
containing the 81-, 47-, 38-, 36-, and 15-kDa proteins were
cross-linked to labeled atpB or psbA leaders in the
presence of a 50- and 100-fold mass excess of competitor RNAs relative
to the labeled probe. If binding of a particular protein to a leader is
a gene-specific event, then unlabeled competitor RNA from a deletion
mutant will not compete the binding of that protein from the labeled
5`-UTR of the gene deleted in the competing mutant strain. Total RNA
from the
psbA strain competes the binding of the 81-,
47-, 38-, and 36-kDa proteins to the atpB leader more strongly
than does
atpB RNA (data not shown). As observed
previously (Fig. 6), the 81-kDa protein is the most efficiently
competed of the three proteins using either
atpB or
psbA RNA. Binding of the 47-kDa protein to the atpB leader was less affected by the presence of 100-fold excess
atpB RNA than any of the other proteins, suggesting one
of the multiple forms of this protein may be specific for this leader.
Presence of excess
atpB or
psbA RNA also
preferentially reduced cross-linking of the 81-kDa protein, and to a
lesser extent the 47-kDa protein, to the psbA leader. Since
these competition experiments were done with pooled column fractions,
we cannot rule out the possibility that dynamic associations between
proteins in different fractions affect the overall UTR binding pattern
of the pooled mixture to a particular chloroplast leader, due to
protein:protein interactions.
Figure 7:
Mapping of the binding sites of the 81-,
47-, and 38-kDa proteins on the atpB and rps7 5`-UTRs
using RNase T1 gel mobility shift assays. The bands indicated (*)
correspond to complexes between the atpB (A) and rps7 (D) 5`-UTRs and a single heparin-Actigel column
fraction from spr-u-1-27-3 (+spec) which contains the
81-, 47-, and 38-kDa binding proteins. Denaturing polyacrylamide gel
analysis of the RNA component of the RNase T1 protected complexes for atpB (B, lane 2) and rps7 (E, lane 2). RNA size markers generated by
complete digestion of P-labeled 5`-UTRs of atpB (B, lane 1) and rps7 (E, lane 1) are depicted. RNase T1 protected fragments (
)
are aligned below their respective 5`-UTR sequences with the putative
SD sequences located above (C and F). Secondary
structures predicted by the Zuker m-fold algorithm for the atpB (C) and rps7 (F) 5`-UTRs are shown. The
RNA sequences protected by the 81-, 47-, and 38-kDa proteins (B and E, lanes 2) are shown in bold on
the secondary structures for atpB (C) and rps7 (F).
To identify the RNA protected by the 81-, 47-, and 38-kDa proteins, full-length transcripts of the atpB and rps7 5`-UTRs were digested to completion with RNase T1, and the fragments were resolved by denaturing PAGE (Fig. 7, B and E, lane 1). Analysis of the RNase T1-protected sequences for both the atpB and rps7 leaders on the same denaturing gel resolved several fragments for each leader (Fig. 7, B and E, lane 2) which were resistant to further hydrolysis by RNase T1 (data not shown). Protected fragments of 35, 34, and 22 nucleotides with longer exposure were resolved for the atpB 5`-UTR (Fig. 7, B, lane 2). Protected fragments of 30, 28, 20, and 19 nucleotides were detected for the rps7 leader (Fig. 7, E, lane 2). Alignment of the atpB and rps7 fragments protected from RNase T1 digestion on the primary sequence and the putative secondary structures predicted by the Zuker m-fold algorithm (44) are shown in Fig. 7, C and D. Due to the high A + T content of these leaders they probably fold into a variety of conformations with the native state of the mRNA being an equilibrium mixture of many different conformations. Further experiments will be necessary to determine the specific structures of these leaders as well as the specific binding sites for the individual trans-acting factors.
Our aim has been to characterize proteins present in cells of C. reinhardtii grown under different environmental conditions
that bind to the 5`-UTRs of chloroplast mRNAs and affect their
translation. In this way we hoped to identify proteins that interact
with all chloroplast mRNAs (core proteins) and those which might be
specific for a group of genes with related functions (e.g. chloroplast protein synthesis versus photosynthesis) or
for cells grown in a specific environment (light versus dark,
acetate versus CO). Previous studies of proteins
that regulate translation of chloroplast mRNAs in this alga have
focused strictly on trans-acting regulatory proteins required for
expression of specific chloroplast genes(1, 45) .
In C. reinhardtii and other chlorophytes, transcription of nuclear-encoded
chloroplast proteins involved in light harvesting and carbon fixation
appear to be repressed by
acetate(13, 16, 42) . We find that
mixotrophically grown cells of C. reinhardtii accumulate
2-fold more D1 protein than phototrophically grown cells, with no
change in the level of accumulation of the psbA mRNA (Fig. 1). Elevated levels of the D1 protein in mixotrophically
grown cells may result from decreased turnover in response to the
reduction in the rate of O evolution observed compared to
that of phototrophically grown cells(48) . In contrast, LSU
levels decrease 2-fold in cultures grown in the light or dark in the
presence of acetate as a carbon source compared to phototrophically
grown cells. While accumulation of both rbcL and atpB messages was reduced under mixotrophic conditions, only the level
of LSU protein accumulation was coordinately reduced and
subunit
protein remained virtually constant (Fig. 1). Clearly the
expression of these key chloroplast genes is largely being modulated
post transcriptionally in response to carbon source. In contrast,
accumulation of r-proteins S7 and S12 and their respective mRNAs are
much less affected by carbon source or illumination during growth.
Experiments in which the unlabeled 5`-UTR of atpB or rbcL was competed against each of the labeled 5`-UTRs (data
shown in Fig. 6for only rps12) strongly suggest that
the same 81- and 47-kDa proteins present in the particular column
fraction analyzed bind to all leaders. However, since 81- and 47-kDa
species are frequently found in more than one fraction and the 47-kDa
protein clearly migrates as a doublet in several cases ( Fig. 4and Fig. 5), we cannot rule out the possibility
that several forms of these proteins exist. Indeed experiments in which
total unlabeled RNA from the atpB and
psbA mutants was competed against the labeled atpB and psbA leaders strongly suggest the existence of at least two
47-kDa species, one of which is specific for atpB and the
other for psbA (Fig. 7). Two other proteins may also
occur in modified forms. The 56-kDa protein present in spr-u-1-27-3 (-spec) extracts which binds to the rps12 leader
elutes in a single high salt fraction compared to elution of the same
protein over three fractions in other extracts (Fig. 5). The
15-kDa protein which elutes in a low salt fraction in most extracts
occurs in a single high salt fraction binding to the rbcL leader in extracts of spr-u-1-27-3 (-spec) (data
not shown).
In extracts from cells with reduced chloroplast protein synthesis (ac-20 cr-1 and spr-u-1-27-3 +spec), which preferentially translate mRNAs for chloroplast encoded r-proteins (9, 18, 49) , a 36-kDa protein present in wild type cells is missing or greatly reduced. This suggests that the 36-kDa protein could be synthesized on chloroplast ribosomes or required for translation of photosynthetic protein mRNAs. The relative enrichment of the 62-kDa protein relative to the 81- and 47-kDa proteins in the extracts from ac-20 cr-1 (Fig. 5) may be related to the permanent deficiency of chloroplast ribosomes in this mutant strain.
The several differences observed in the binding patterns of these proteins between leaders of specific r-protein and photosynthetic protein mRNAs will require additional study before they can be interpreted in terms of the hierarchical model for control of translational regulation in chloroplasts that we postulated recently (1) . Based on our current observations, one can postulate that the ubiquitous 81- and 47-kDa proteins (or a subset of their various forms), may serve to mark the chloroplast mRNAs for translation. Association of other proteins with this complex in a gene or class-specific fashion(1) , may yield a complex competent for translation initiation.
In conclusion, our results emphasize the importance of defining the 5`-UTR-binding proteins present in different cell extracts and their spectrum of interactions with the 5`-UTRs of different chloroplast genes. Either cloned gene(s) or antibody probes will be necessary to resolve the conundrum that 46-47-kDa trans-acting proteins have been reported to be specific for regulating expression of the chloroplast psbA(5, 6, 7) or psbC(8) genes, whereas our data suggest that one or more 47-kDa species bind ubiquitously to all chloroplast leaders examined. Only in this way can trans-acting factors that bind generally to chloroplast 5`-UTRs be distinguished from the putative gene specific factors that have so far been characterized(1, 4) .