The chloroplast ribosome comes of age. The Translational Apparatus of Photosynthetic Organelles: a NATO Advanced Research Workshop, Grenoble, France, July 3-6, 1990.
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Animals↗
Biomedical subjects
R Mache
Publications and source records attributed to R Mache.
Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein.
A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast. The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma. Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome. Clones were isolated from a cDNA library constructed in the expression vector lambda gt11, using specific antibodies raised against the CS-S5 protein. A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation. This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts.
Amino Acid Sequence↗
Cotranscription of the S10- and spc-like operons in spinach chloroplasts and identification of three of their gene products.
The organisation and expression of the rpl22, rps3, rpl16 and rpl14 genes, which belong to the S10- and spc-like operons of spinach chloroplasts, have been studied. Northern experiments and nuclease S1 mapping show that the two operon-like groups of genes are cotranscribed. It is demonstrated that the intron-containing rpl16 gene is spliced in vivo. Based on amino acid composition and protein sequence data, the products of the rpl22, rpl16 and rpl14 genes are identified respectively as the spinach chloroplast ribosomal proteins CS-L13, CS-L24 and CS-L29. The rpl22 gene product is a 5S rRNA binding protein and therefore is distinguishable from the homologous Escherichia coli L22 ribosomal protein.
Amino Acid Sequence↗
Higher order structure of chloroplastic 5S ribosomal RNA from spinach.
The secondary and tertiary structure of chloroplastic 5S ribosomal RNA from spinach was investigated by the use of several chemical and enzymatic structure probes. The four bases were monitored at one of their Watson-Crick base-pairing positions with dimethyl sulfate [at A(N1) and C(N3)] and with 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate [at G(N1) and U(N3)]. Position N7 of purines was probed with diethyl pyrocarbonate (adenines) and with dimethyl sulfate (guanines). Ethylnitrosourea was used to probe phosphate involved in tertiary interaction or in cation coordination. In order to estimate the degree of stability of helices, the various chemical reagents were employed under "native" conditions (300 mM KCl and 20 mM magnesium at 37 degrees C), under "semidenaturing" conditions [1 mM ethylenediaminetetraacetic acid (EDTA) at 37 degrees C], and under denaturing conditions (1 mM EDTA at 90 degrees C). Unstructured regions were also tested with single-strand-specific nucleases T1, U2, and S1 and double-stranded or stacked regions with RNase V1 from cobra Naja naja oxiana venom. The results confirm the existence of the five helices and the two external loops proposed in the consensus model of 5S rRNA. However, the regions depicted as unpaired internal loops appear to be folded into a more complex conformation. A three-dimensional model derived from the present data and graphic modeling for a region encompassing helix IV, helix V, loop D, and loop E (nucleotides 70-110) is proposed. Nucleotides in the so-called loop E (73-79/100-106) display unusual features: Noncanonical base pairs (A-A and A-G) are formed, and three nucleotides (C75, U78, and U105) are bulging out. This region adopts an unwound and extended conformation that can be well suited for tertiary interactions or for protein binding. Several bases and phosphates candidate for the tertiary folding of the RNA were also identified.
Base Composition↗
Expression of the rpl23, rpl2 and rps19 genes in spinach chloroplasts.
The expression of the spinach rpl23, rpl2 and rps19 chloroplast genes has been studied. The rpl23 gene identified in tobacco and Marchantia, is split into two overlapping reading frames in spinach. S1 mapping has shown that initiation sites could occur upstream of each reading frames. A large transcription unit is also present covering the rpl2 and rps19 genes. The rps19 and rpl2 gene products are identified by NH2-terminal amino acid sequences. They correspond to spinach chloroplast ribosomal proteins CS-S23 and CS-L4, respectively. No product of the rpl23 gene was detected in the chloroplast 50S ribosomal subunit. This strongly suggest that a corresponding gene has been transfered into the nucleus.
Amino Acid Sequence↗
Characterization of a large inversion in the spinach chloroplast genome relative to Marchantia: a possible transposon-mediated origin.
A 7,022 bp BamHI-EcoRI fragment, located in the inverted repeat of spinach chloroplast, has been sequenced. It contains a 2131 codon open reading frame (ORF) homologous to both tobacco ORFs 581 and 1708, and to Marchantia ORF 2136. Relative to the Marchantia chloroplast genome, spinach ORF 2131 is located at the end of a large inversion; the other end point is close to trnL, the position of which is the same in Marchantia, tobacco and spinach. In Marchantia, two 8 bp direct repeats flanking two 10 bp indirect repeats are present near the end points of the inversion. These repeats may result from a transposon-mediated insertion which would have facilitated the subsequent inversion. From a comparison of the gene organization of the spinach, tobacco, and Marchantia genomes in this region, we propose a step-wise process to explain the expansion of the inverted repeat from a Marchantia-like genome to the spinach/tobacco genome.
Base Sequence↗
Presence of a 16S rRNA pseudogene in the bi-molecular plastid genome of the primitive brown alga Pylaiella littoralis. Evolutionary implications.
The plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm. is composed of two different circular DNA molecules: the largest carries two rrn operons, and the smallest, only one copy of both 16S and 23S rDNAs. 16S rDNA copies located on both molecules have been cloned and their nucleotide sequences determined: they are 65% homologous to one another. The expression of these genes was assayed by hybridizing in vivo labelled P. littoralis rRNAs to both clones, and specific oligonucleotides to total RNA from P. littoralis. Results indicate that the 16S rDNA copy located on the small molecule is a pseudogene. Comparisons of the functional gene with other 16S rRNA genes shows that chloroplasts from green plants emerged earlier from the cyanobacterial lineage than Euglena gracilis and Pylaiella littoralis plastids.
Base Sequence↗
Phosphorylation in vitro of the large subunit of the ribulose-1,5-bisphosphate carboxylase and of the glyceraldehyde-3-phosphate dehydrogenase.
A protein kinase activity responsible for the in vitro phosphorylation of at least six endogenous polypeptides including the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is present in the stroma (3000 X g supernatant, S30) of spinach chloroplasts. The phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is strongly enhanced when sodium fluorure is used as a protein phosphatase inhibitor. Phosphorylation occurs on threonine and serine residues. The protein kinase involved is not Ca2+-dependent. There is also evidence for a protein phosphatase activity which suggests a coupled regulation by a phosphorylation-dephosphorylation process. The phosphorylating activity is drastically reduced when S30 is prepared from leaves harvested after a dark period. Phosphorylation of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit is not related to its own synthesis. The in vitro phosphorylation of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13) is also demonstrated.
Chloroplasts↗
Nucleotide sequence of the spinach chloroplast tRNA(CAALeu) gene and its flanking regions.
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Base Sequence↗
Structure and transcription of the 5S rRNA gene from spinach chloroplasts.
The nucleotide sequence of the spinach chloroplast 5S rRNA gene and its flanking regions has been determined. A prokaryotic type promoter is to be found upstream of the 5S rRNA gene. Northern blot experiments with selected gene probes show that the 5S gene is co-transcribed with the other ribosomal genes of the operon. This result is confirmed by 5' S1 mapping of in vivo RNAs synthesised in chloroplasts or in an E. coli strain harboring a multicopy plasmid containing the 5S rRNA gene and its flanking regions. In vitro transcription experiments show that initiation of transcription does not occur at the level of the putative 5S rRNA gene promoter. Therefore, we conclude that the 5S rRNA is synthesized only be co-transcription of its gene with the other ribosomal genes of the operon. 3' S1 nuclease mapping in the spacer region between the 4.5S and the 5S rRNA genes reveals a set of protected fragments located in an A.T rich region downstream of a very stable hairpin and immediately upstream of the putative 5S promoter. This result is interpreted by the presence of preterminated transcripts or processing sites in this region.
Base Sequence↗
Nucleotide sequence of the spinach chloroplast 4.5S ribosomal RNA gene and of its 5' flanking region including the 3' end of the 23S rRNA gene.
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Base Sequence↗
A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L.
Ribosomal subunits are isolated from potato tuber mitochondria devoid of contaminating organelles. The sedimentation constants of the two mitochondrial ribosomal subunits are 33S and 50S respectively. The apparent sizes of the high molecular weight RNAs are 19S and 25S. The proteins of these ribosomes have been analyzed by two-dimensional electrophoresis in SDS polyacrylamide gels to determine their number and molecular weights. The small subunit contains 35 protein species ranging from 8 to 60 kDa. The 50S large subunit contains 33 protein species ranging from 12 to 46 kDa. These preliminary results are the first analysis made on mitochondrial ribosomes from a higher plant.
Mitochondria↗
In vitro transcription initiation of the spinach chloroplast 16S rRNA gene at two tandem promoters.
Two potential prokaryotic promoters, P1 and P2, are characterized 164 and 114 bp upstream of the spinach chloroplast 16S rRNA gene. The strengths of these promoters, calculated according to an homology score established for E. coli RNA-polymerase, are identical. Experiments performed with a Taq I-DNA fragment, containing 16 bp of the 16S rDNA and 243 bp upstream of the gene, give evidence that in vitro, E. coli RNA-polymerase starts transcription at these two promoters. These results are based on both the size of the transcripts and their nucleotide sequences. A possible regulation by differential control of these dual promoters is suggested. S1 mapping with RNAs extracted either from green or from etiolated spinach plants, indicates that, at these two steps of plastid development, transcription in vivo starts at P1. Surprisingly only P2 appears to be conserved in the homologous sequences reported for maize, mustard and Spirodela.
Chloroplasts↗
Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose.
Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20 degrees C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.
Journal Article↗
In organello and in vitro phosphorylation of chloroplast ribosomal proteins.
Two chloroplast ribosomal proteins are phosphorylated in isolated chloroplast. One in the large subunit (L18) and one in the small subunit ( LS31 ). The phosphorylation is light dependent and occurs on a serine residue for both ribosomal proteins. These two proteins and other chloroplast ribosomal proteins are also phosphorylated in vitro using [gamma 32P]-ATP and a cAMP -dependent or a cAMP - independent protein kinase. The existence of a protein-kinase bound to chloroplast 70S ribosomes is also demonstrated, the enzyme is able to phosphorylate almost every chloroplast ribosomal protein.
Autoradiography↗
Comparison of ribosomal proteins of chloroplast from spinach and of E. coli.
A comparison of ribosomal proteins from Escherichia coli and from chloroplasts of Spinach was made using two separate methods: electrophoretic migration and immunochemical cross-reaction between blotted E. coli ribosomal proteins and chloroplast ribosomal subunits antisera. It is shown that L2 from E. coli (E-L2) and L4 from chloroplasts (CS-L4) comigrated and that E-L2 immunologically cross-reacted with the isolated CS-L4 antibody. Co-migration was observed for three additional couples of 50S ribosomal proteins. It is also shown that at least one 30S E. coli ribosomal protein immuno-cross reacted with a 30S chloroplast antiserum and that three couples of 30S ribosomal proteins comigrated.
Antigen-Antibody Complex↗
Structure and transcription of the spinach chloroplast rDNA leader region.
A cloned fragment of spinach chloroplast DNA carrying 140 bp of the 16S rRNA gene and 691 bp upstream this gene has been analysed by DNA sequencing, by in vitro transcription, by S1 mapping with chloroplast RNAs and purified 16S rRNA from 30S ribosomal subunits. A tRNAVal gene has been located between the position 394 and 465. Crude chloroplast RNA polymerase has been purified by heparin sepharose chromatography of a 80 000 g supernatant from pure lysed spinach plastids and used to transcribe the cloned Bg1 II-Pvu II DNA fragment. Four in vitro transcripts of about 830, 550, 350 and 260 bases were obtained whatever RNA polymerase used: the chloroplast or the E. coli enzyme. The transcripts of 550 and 260 bases are initiated by ATP. S1 mapping with in vivo chloroplasts RNAs on 5' labelled separated strands from Bg1 II-Pvu II fragments indicates 2 protected DNA fragments respectively of 140 and 260 bases on the strand which codes for rRNAs and possibly one protected DNA fragment of 550 bases on the other strand. The start site of the 260 bases transcript might correspond to the initiation site of transcription of the rRNA genes. The possibility that the 550 bases transcription of the non coding strand for rRNA genes corresponds to the beginning of a mRNA is discussed.
Base Composition↗
Visualization of a Spinach Plastid Transcriptionally Active DNA-Protein Complex in a Highly Condensed Structure.
A transcriptionally active DNA-protein complex isolated from spinach Spinacia oleracea plastids is visualized by electron microscopy in different conditions. This structure, after glutaraldehyde fixation, is highly condensed. DNA is supertwisted with proteins bound to it producing a beaded substructure. When glutaraldehyde fixation is omitted this structure is less condensed and DNA fibrils come out from a proteinous central body. The DNA-protein complex can be separated into two populations by CsCl centrifugation: one with a buoyant density of 1.570 grams per cubic centimeter and the other of 1.610 grams per cubic centimeter. By visualization of these two populations, it is concluded that proteins are either firmly bound to DNA in the central body, or more loosely bound to the DNA fibrils. These latter proteins could play a role in enzymic functions and/or in the supercoiling of DNA.The DNA from the DNA-protein complex possesses all fragments that belong to pure circular chloroplast DNA hydrolyzed by two restriction enzymes: Bam HI and Eco RI. Some molecules observed in a supercondensed form with a beaded substructure probably contain entire chloroplast DNA molecules.A hydrolysis test with microccocal nuclease gives no indication of the presence of ;nucleosome-like' structures. Thirty-six polypeptides with molecular weights ranging from 12,000 to 180,000 are present in the complex, and seven of them are highly soluble in 0.4 n H(2)SO(4); their molecular weights range from 14,000 to 46,000 as shown by two-dimensional gel electrophoresis.No linolenic acid can be detected in the preparation, indicating the absence of chloroplast membranes.
Journal Article↗