Chloroplast Ribosomes Research Articles

Evidence that the fragmented ribosomal RNAs of Chlamydomonas mitochondria are associated with ribosomes

The discontinuous and scrambled organization of the small subunit and large subunit rRNA coding regions in Chlamydomonas mitochondrial DNA has been well documented. Our goals were to demonstrate that the small transcripts produced by these coding regions in Chlamydomonas eugametos are assembled into mitochondrial ribosomes and to characterize the sedimentation properties of these ribosomes and their subunits in sucrose gradients. Putative mitochondrial ribosomes (60–66S) and their large (44–50S) and small (35–39S) subunits were identified by slot blot hybridization which sedimented independently of the chloroplast and cytosolic ribosomes. A crude mitochondrial pellet prepared from C. eugametos was enriched for mitochondrial small subunit and large subunit rRNA subfragments thereby providing independent confirmation of the mitochondrial association of these rRNA molecules.

Ultrastructural changes in aging leaves of a light‐grown achlorophyllous mutant of barley

The pattern and sequence of cellular degradation during the course of leaf senescence remains obscure and the nature of the trigger that induces cell senescence is unknown. In order to probe the pre‐mortem phase of senescence temporal changes in cell ultrastucture were studied in aging leaves of light‐grown achlorophyllous Hordeum vulgare L. cv. Dyan mutant seedlings. Electron microscope examination of the ultrastructure of mesophyll cell plastids revealed the absence of ribosomes and a highly disorganized prolamellar body. Both the number and size of plastoglobuli increased with aging and this change coincided with depletion of starch grains and dilation of lamellar membranes. Aging of mesophyll cells occurred coincident with a decline in ribosome content of the cytoplasm and loss of matrix granularity. Loss of ribosomes associated with the outer nuclear envelope membrane and a reduction in chromatin were also apparent. Only after 10 days was there evidence of loss of internal membrane integrity and swelling of mitochondrial cristae. Compartmentation was thus maintained during the aging process with membrane dissolution occurring late in senescence. These results suggest that an inability to produce chlorophyll and carotenoids and form thylakoid stacks due to the absence of plastid ribosomes, contributes to the rapid onset of senescence in light‐grown achlorophyllous seedlings. Furthermore, disruption of chloroplast ribosome synthesis/assembly may constitute part of the plastid signal involved in triggering cell senescence.

Chloroplast ribosomes and protein synthesis

Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival.

Inefficient rpl2 splicing in barley mutants with ribosome-deficient plastids.

Analysis of transcript accumulation and splicing in plastids of four nuclear mutants of barley revealed that the ribosomal protein L2 (rpl2) gene transcripts containing a group II intron remained entirely unspliced, whereas the intron of the ribosomal protein L16 (rpl16) gene (linked with the rpl2 gene in the same operon) was removed in the mutant plastids. Also, the transcripts of other genes containing group II introns (ribosomal protein S16 gene, rps16; NADH dehydrogenase ND2 gene, ndhB; cytochrome f gene, petD; and intron-containing reading frame 170, irf170) and of the tRNA for leucine, trnL (UAA), possessing the only chloroplast group I intron, were found to be spliced. The mutants used in this investigation are considered to be nonallelic; this excludes the possibility that a single nuclear gene is responsible for the impaired splicing of rpl2 transcripts. The mutants, however, have a severe deficiency in chloroplast ribosomes in common; this deficiency is evident from the lack of the essential ribosomal protein L2 and from an extremely low steady state level of plastid rRNAs. From these results, we conclude that a functioning translational apparatus of the organelle is a prerequisite for splicing of the chloroplast rpl2 class II intron but not for splicing of at least five other group II intron-containing transcripts. This provides genetic evidence for a chloroplast DNA-encoded component (e.g., a maturase) involved in the splicing of rpl2 pre-mRNA.

Recognition of novel and divergent higher plant chloroplast ribosomal proteins by Escherichia coli ribosome during in vivo assembly.

Architecture of higher plant chloroplast ribosomes involves additional protein domains over that found in the Escherichia coli ribosome, although the rRNAs in these two kinds of ribosomes are very similar in length and sequence (Subramanian, A. R. (1993) Trends Biochem. Sci. 18, 177-180). Here, we show that two chloroplast-specific protein domains (a novel chloroplast ribosomal protein of the 30 S subunit, called Psrp-1 or S22, and a divergent protein of the 50 S subunit with long terminal extensions and low homology to its E. coli counterpart, L21) are both incorporated in E. coli ribosomes and polysomes when their gene constructs are expressed in E. coli. Also, the 67-residue NH2-terminal extension in chloroplast L21 by itself is incorporated. Thus, our results indicate preexisting binding sites for novel chloroplast-specific ribosomal proteins/domains on eubacterial ribosomes. Additionally, we observed cleavage of the chloroplast-targeting transit peptide (present in the expressed Psrp-1 precursor), indicating protease(s) of the required specificity in E. coli cells. The expression of chloroplast L21 with its NH2-terminal extension was inhibitory to E. coli growth, suggesting a drastic effect of the latter on some property of L21. Expression of Psrp-1 was neutral, consistent with a function only in chloroplast translation. Based on analysis of the assembly of Psrp-1 and various L21 fragments in E. coli ribosomes, a general model for studying ribosomal protein-ribosome interactions is suggested.

Protein Substitution in Chloroplast Ribosome Evolution: A Eukaryotic Cytosolic Protein has Replaced its Organelle Homologue (L23) in Spinach

The chloroplast translational system differs from the eubacterial ones in containing several ribosomal proteins (RPs) that have no apparent homologues in eubacteria, and in having their RP genes distributed in two cellular genome compartments. The genes maintained in the organelle genome encode mainly ribosome assembly proteins. The discovery in spinach and related plants ( Caryophyllidae) of a disrupted chloroplast gene encoding the ribosome assembly protein L23 raised speculations about the transfer of the functional rpl23 gene to the nucleus or the evolutionary loss of L23 protein requirement. To solve this problem, we overexpressed in E. coli the intact rpl23 gene from corn ( Zea mays), purified the protein and raised antibodies. Based on immunoanalysis, we show that a prokaryotic-type L23 protein is absent in spinach. Concomittantly we have isolated a new protein from spinach chloroplast 50 S ribosomal subunits and determined its amino acid sequence. The data revealed an unexpectedly high sequence identity to the eukaryotic family of cytosolic L23 proteins (reported from yeast, trypanosome and rat), with conservation of a peptide motif responsible for the specific interaction of these proteins with domain III of 26 S and 23 S rRNA. We propose that the prokaryotic-type L23 protein in the chloroplast ribosomes of Caryophyllidae has been replaced by a homologue of the eukaryotic cytosolic L23 family. These results represent the first case of a protein (gene) substitution in chloroplast ribosome evolution, and open a new view on how the nuclear genome could progressively exert stronger over the chloroplast translational system. We also describe experiments on the incorporation of chloroplast L23 into E. coli ribosomes, its effect on cell growth, and an unexpected immuno cross-reaction between two chloroplast RP families.

A novel RNA gene in the tobacco plastid genome: its possible role in the maturation of 16S rRNA.

A small plastid-encoded RNA (spRNA, 218 nt) has been detected in tobacco. The corresponding locus (sprA) does not contain any open reading frame and is actively transcribed from its own promoter, as shown by ribonuclease protection assays using in vitro capped RNAs. Gel-shift and UV-crosslinking experiments showed the formation of a specific complex between spRNA and chloroplast polypeptides. The mobility of the complex was further shifted when a transcript bearing part of the 16S rRNA leader sequence was added to the incubation mixture. Glycerol gradient fractionation of a chloroplast lysate indicated a preferential sedimentation of spRNA at 15-20S and 70S. These observations, and the potential base-pairing with the leader sequence of pre-16S rRNA, suggest a role for spRNA in chloroplast ribosome biogenesis, i.e. 16S rRNA maturation. By sequencing of tomato plastid DNA and heterologous northern hybridizations, the presence of sprA homologs and their expression in a number of dicot plants have also been shown.

Multicopy GTPase center protein L12 of Arabidopsis chloroplast ribosome is encoded by a clustered nuclear gene family with the expressed members closely linked to tRNA(Pro) genes.

A conserved architectural feature of ribosomes is a protuberance (the stalk) in the large subunit, essential for ribosomal interactions with translational factors and GTP hydrolysis and generated by two dimers of L12, the only multicopy protein in ribosomes. In higher plants, the rpl12 gene for chloroplast L12 is located in the nucleus. We report here the cloning and sequencing of this nuclear gene from Arabidopsis thaliana, revealing the first gene family for a chloroplast ribosomal protein (RP). A single cluster/haploid genome of three rpl12 genes is located in the sequenced 9.1-kilobase region of the Arabidopsis genome. Two of the rpl12 genes encode identical mature proteins, and the third encodes a 25% divergent RP, although the chloroplast-targeting transit peptide in each is distinct. The rpl12 genes encoding identical RPs are closely linked at their 5' ends to identical cytosolic tRNA(Pro) genes with a < 250-base pair spacer. Reverse transcriptase polymerase chain reaction experiments with total RNA isolated from Arabidopsis (and characterization of several L12 cDNA clones) show that only the tRNA-linked rpl12 genes are expressed. We also show (by polymerase chain reaction experiments with isolated total DNA) that this tRNA(Pro)-rpl12 gene linkage is conserved in spinach (inferred to contain a single gene copy) indicating its importance. The previously described enhanced translation of spinach L12 mRNA from its two tandem AUG codons and the two functional rpl12 genes in Arabidopsis probably provide two mechanisms for generating the four copies of L12/chloroplast ribosome, qualitatively different from those attempted in eubacteria.

Effect of the secondary structure in the Euglena gracilis chloroplast ribulose-bisphosphate carboxylase/oxygenase messenger RNA on translational initiation.

The results reported in the previous paper indicate that the translational start site of the Euglena gracilis chloroplast mRNA for the large subunit of ribulose-bisphosphate carboxylase/oxygenase (rbcL) is not defined by primary sequence elements (Koo, J.S., and Spremulli, L.L. (1994) J. Biol. Chem. 269, 7494-7500). In the work presented here, the effects of secondary structure in the 5'-untranslated leader of the rbcL mRNA have been examined. Only weak secondary structure can be detected in the 5'-untranslated leader of the rbcL message by enzymatic and computer analysis. Further reduction of the weak secondary structure of this message by site-directed mutagenesis does not significantly affect the ability of this message to participate in initiation complex formation. The secondary structure near the translational start site was increased by the introduction of an inverted repeat sequence and by site-directed mutagenesis. Messages with increased secondary structure are much less active in initiation complex formation if the structural element introduced is within approximately 10 nucleotides of the start codon. These results suggest that the translational start site in this chloroplast mRNA is specified by the presence of an AUG codon in an unstructured or weakly structured region of the mRNA. No specific sequences around the start codon, either upstream or immediately downstream, were found to have important information directing the chloroplast ribosome to the start site of this mRNA.

The photosynthetic endosymbiont in cryptomonad cells produces both chloroplast and cytoplasmic-type ribosomes

Cryptomonad algae contain a photosynthetic, eukaryotic endosymbiont. The endosymbiont is much reduced but retains a small nucleus. DNA from this endosymbiont nucleus encodes rRNAs, and it is presumed that these rRNAs are incorporated into ribosomes. Surrounding the endosymbiont nucleus is a small volume of cytoplasm proposed to be the vestigial cytoplasm of the endosymbiont. If this compartment is indeed the endosymbiont's cytoplasm, it would be expected to contain ribosomes with components encoded by the endosymbiont nucleus. In this paper, we used in situ hybridization to localize rRNAs encoded by the endosymbiont nucleus of the cryptomonad alga, Cryptomonas phi. Transcripts of the endosymbiont rRNA gene were observed within the endosymbiont nucleus, and in the compartment thought to represent the endosymbiont's cytoplasm. These results indicate that the endosymbiont produces its own set of cytoplasmic translation machinery. We also localized transcripts of the host nucleus rRNA gene. These transcripts were found in the nucleolus of the host nucleus, and throughout the host cytoplasm, but never in the endosymbiont compartment. Our rRNA localizations indicate that the cryptomonad cell produces two different of sets of cytoplasmic-type ribosomes in two separate subcellular compartments. The results suggest that there is no exchange of rRNAs between these compartments. We also used the probe specific for the endosymbiont rRNA gene to identify chromosomes from the endosymbiont nucleus in pulsed field gel electrophoresis. Like other cryptomonads, the endosymbiont nucleus of Cryptomonas phi contains three small chromosomes.

Chloroplast ribosomes and protein synthesis.

Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival.

Spectinomycin interacts specifically with the residues G1064and C1192in 16S rRNA, thereby potentially freezing this molecule into an inactive conformation

The upper stem of helix 34, consisting of the base-paired sequences C1063G1064U1065 and A1191C1192G1193, is suggested to be involved in the binding of spectinomycin. In E. coli 16S rRNA, each of the three mutations at position C1192 confers resistance to spectinomycin. In chloroplast ribosomes from tobacco plants and algae, resistance is conferred by single mutations at positions 1064, 1191, and 1193 (E. coli numbering). Since each of these mutations disrupt any of the three basepairs in the upper stem of helix 34, it has been postulated that spectinomycin can bind to this region and inhibit protein synthesis, only if its nucleotides are basepaired. We have tested this hypothesis by introducing disruptive and compensatory mutations that alter the basepair G1064-C1192. Using the specialized ribosome system, the translational activity of such mutants was determined, in the absence and presence of spectinomycin. We show that any of the three disruptive mutations A1064, C1064, and U1064 confer resistance, in accordance with the model for spectinomycin binding. Compensatory mutations A1064U1192, C1064G1192, and U1064A1192, however, maintained the resistance. This indicates that a basepaired conformation as such is not sufficient for spectinomycin binding, but rather that a G-C pair at positions 1064 and 1192 is required. In addition, we find that the translational activity of specialized ribosomes containing the mutations C1064G1192 is 5-fold lower compared to that of ribosomes containing any of the other mutations introduced, regardless whether spectinomycin is present or not. Since the introduction of C1064G1192 is expected to increase the stability of the upper stem of helix 34, we suggest that these mutations impair ribosome function by preventing the (transient) disruption of the upper stem. By analogy, we speculate that spectinomycin blocks protein synthesis by stabilizing the upper stem. In both cases, the 30S subunit would be frozen into an inactive conformation.

The Onset of 70S Chloroplast Ribosome Formation is Determined by an Early Heat-Sensitive Stage in the Ontogeny of Rye Leaves

The formation of plastidic 70S ribosomes can be specifically eliminated in developing leaves of rye (Secale cereale L.) when seedlings are continuously grown at an elevated temperature of 32°C. Labeling experiments with [35S]methionine and [3H]uridine have indicated that in fully differentiated leaf tissue grown under permissive conditions (22°C) exposure to 32°C did not impair current 70S ribosome biosynthesis. Neither the synthesis or processing of plastidic rRNA nor the synthesis and integration of proteins into 70S ribosomes were directly affected. However, when the leaves were grown at 32°C, the young tissue of the basal growing zone that had been newly formed during a 24 h or longer heat exposure appeared to become unable to assemble and accumulate 70S ribosomes, whereas all ribosomal constituents that were examined were still present. Complete plastid ribosomes were thus progressively diluted out. This heat-sensitivity indicates the existence of an early step in the differentiation of leaf cells which controls the expression of some limiting factor for plastid ribosome formation or assembly. The expression of plastid ribosome constituents was not tightly coregulated. Only after prolonged and extensive depletion of 70S ribosomes were plastidic transcripts (rRNA) diminished and several ribosomal proteins lost. Several nuclear-encoded ribosomal proteins and the transcript for the nuclear-encoded protein L12 were maintained at high levels in 70S ribosome-deficient leaf sections.

A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11

We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis. The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type. Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C. reinhardtii. Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase. Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time. L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit. Thiostrepton-resistant mutants of Bacillus megaterium and B. subtilis lack L11, show reduced ribosome activity, and have slow growth rates. Similarities between the thiostrepton-resistant mutants of bacteria and C. reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E. coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.

Identification of the nuclear-encoded chloroplast ribosomal protein L12 of the monocotyledonous plant Secale cereale and sequencing of two different cDNAs with strong codon bias

Two different cDNA clones (SCL12-1 and SCL12-2) encoding precursors of a chloroplast ribosomal protein with homology to L12 from Escherichia coli were isolated from rye leaf cDNA libraries and sequenced. The corresponding polypeptide of rye chloroplast ribosomes was identified. The sequences for the mature proteins of M r 13447 and 13609 share 85% amino acid identity. The mature polypeptide of clone SCL12-1 has an amino acid identity of 71%, 72% or 44%, respectively, relative to L12 proteins from spinach, tobacco, or E. coli. Codon usage of the rye L12 cDNAs shows a high preference (97% and 82%) for G or C in the third base position.

Complete sequence of Euglena gracilis chloroplast DNA.

We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.

Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis.

Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced. These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278). The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively. The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively. An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein. Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins. The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein. Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA. The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.

Tobacco chloroplast ribosomes contain a homologue of E. coli ribosomal protein L28

The genes ror ribosomal proteins L28 and L33 constitute an operon ( rpmBG) in E. coli, but in plant chloroplasts L33 is encoded by the chloroplast DNA and L28 seems to be encoded by the nuclear genome. A 15 kDa protein was isolated from the 50 S subunit of tobacco chloroplast ribosomes and its N-terminal amino acid sequence was determined. A cDNA for this protein was cloned and analyzed. The cDNA encodes a 151 amino acid protein consisting of a predicted transit-peptide of 74 amino acids and a mature protein of 77 amino acids. The mature protein is homologous to E. coli L28, hence we named it chloroplast L28 (CL28). This is the first report on the presence of an E. coli L28-like protein in another organism.

The primary structure of rat ribosomal protein L3

The amino acid sequence of the rat 60S ribosomal subunit protein L3 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L3 has 403 amino acids and has a molecular weight of 46,106. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 7 to 9 copies of the L3 gene. The mRNA for the protein is about 1,400 nucleotides in length. Rat L3 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.

The primary structure of rat ribosomal protein L8

The amino acid sequence of the rat 60S ribosomal subunit protein L8 wad deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L8 has 257 amino acids and has a molecular weight of 28,007. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 4 or 5 copies of the L8 gene. The mRNA for the protein is about 950 nucleotides in length. Rat L8 is homologous to ribosomal proteins from other eukaryotes and to proteins from eubacterial, archaebacterial, and chloroplast ribosomes.