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) .

MATERIALS AND METHODS

Strains and Media

Nucleic Acid Manipulations

()

Northern Analysis

Antisera

Protein Isolation and Immunoblotting

In Vitro RNA/Protein Binding Experiments

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.

Filter Binding Assay

Mapping of Protein Binding Sites

Digitization and Quantification of Autoradiographs

RESULTS

Effects of Different Environmental and Physiological Conditions on the Accumulation of Representative Chloroplast Proteins and Their mRNAs

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) .

In Vitro Gel Retardation and UV Cross-linking Analysis of the 5`-UTR-binding Proteins

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.

Spectrum of 5`-UTR Binding Proteins Present in Cells Grown under Different Environmental Conditions

Effects of Illumination on the Presence of UTR Binding Proteins in Wild Type

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.

Effects of Carbon Source on the Presence of UTR Binding Proteins in Wild Type

UTR Binding Proteins Present under Conditions of Reduced Chloroplast Protein Synthesis

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.

Competition Experiments Demonstrate That the Same Trans-acting Proteins Bind to the 5`-UTRs of Several Different Chloroplast mRNAs

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.

Mapping of the Binding Site(s) for the 47- and 81-kDa Proteins to the 5`-UTRs of atpB and rps7 mRNAs

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.

DISCUSSION

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) .

Environmental Effects on Organelle Gene Expression in Chlamydomonas

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.

Effects of Reduced Levels of Chloroplast Protein Synthesis on Organelle Gene Expression

5`-UTR Binding Proteins Present in Extracts of Cells Grown under Varying Physiological Conditions

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.

Identification of Cis-acting Sequences

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) .

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant GM-19427 (to J. E. B. and N. W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by National Institutes of Health Postdoctoral Fellowship GM-14046 for part of this research.

¶

To whom correspondence should be addressed: Developmental, Cell and Molecular Biology Group, B330G LSRC Bldg., Research Dr., Box 91000, Duke University, Durham, NC 27708-1000. Tel.: 919-613-8157; Fax: 919-613-8177; jboynton{at}acpub.duke.eduuke.edu.

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The abbreviations used are: UTR, untranslated region; r-protein, ribosomal protein; spec, spectinomycin; Rubisco, ribulose-bisphosphate carboxylase/oxygenase; SD, Shine-Dalgarno; SSU, small subunit; LSU, large subunit; PAR, photosynthetically active radiation; HS, high salt; HSA, high salt acetate.

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B. Randolph-Anderson, unpublished results.

ACKNOWLEDGEMENTS

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