The cytochrome b(6)f complex: structural studies and comparison with the bc(1) complex.
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Electron crystallography of the chloroplastic b(6)f complex allowed the calculation of projection maps of crystals negatively stained or embedded in glucose. This gives insights into the overall structure of the extra- and transmembrane domains of the complex. A comparison with the structure of the bc(1) complex, the mitochondrial homologue of the b(6)f complex, suggests that the transmembrane domains of the two complexes are very similar, confirming the structural homology deduced from sequence analysis. On the other hand, the extramembrane organisation of the c-type cytochrome and of the Rieske protein seems quite different. Nevertheless, the same type of movement of the Rieske protein is observed in the b(6)f as in the bc(1) complex upon the binding of the quinol analogue stigmatellin. Crystallographic data also suggest movements in the transmembrane domains of the b(6)f complex, which would be specific of the b(6)f complex. (+info)
cis- and trans-Acting determinants for translation of psbD mRNA in Chlamydomonas reinhardtii.
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Chloroplast translation is mediated by nucleus-encoded factors that interact with distinct cis-acting RNA elements. A U-rich sequence within the 5' untranslated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linking assays, we have identified a 40-kDa RNA binding protein, which binds to the wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor. The functions of several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic activity and the capability to bind RNA by the 40-kDa protein was observed. Furthermore, the data obtained suggest that the poly(U) region serves as a molecular spacer between two previously characterized cis-acting elements, which are involved in RNA stabilization and translation. RNA-protein complex formation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts directly with this Nac2 complex and, as a result, links processes of chloroplast RNA metabolism and translation. (+info)
Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase.
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The molecular mechanism(s) responsible for posttranscriptional gene silencing and RNA interference remain poorly understood. We have cloned a gene (Mut6) from the unicellular green alga Chlamydomonas reinhardtii that is required for the silencing of a transgene and two transposon families. Mut6 encodes a protein that is highly homologous to RNA helicases of the DEAH-box family. This protein is necessary for the degradation of certain aberrant RNAs, such as improperly processed transcripts, which are often produced by transposons and some transgenes. (+info)
Chlorophyll b expressed in Cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins.
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Photosynthetic pigments bind to their specific proteins to form pigment-protein complexes. To investigate the pigment-binding activities of the proteins, chlorophyll b was for introduced the first time to a cyanobacterium that did not synthesize that pigment, and expression of its function in the native pigment-protein complex of cyanobacterium was confirmed by energy transfer. Arabidopsis CAO (chlorophyll a oxygenase) cDNA was introduced into the genome of Synechocystis sp. PCC6803. The transformant cells accumulated chlorophyll b, with the chlorophyll b content being in the range of 1.4 to 10.6% of the total chlorophyll depending on the growth phase. Polyacrylamide gel electrophoresis analysis of the chlorophyll-protein complexes of transformant cells showed that chlorophyll b was incorporated preferentially into the P700-chlorophyll a-protein complex (CP1). Furthermore, chlorophyll b in CP1 transferred light energy to chlorophyll a, indicating a functional transformation. We also found that CP1 of Chlamydomonas reinhardtii, believed to be a chlorophyll a protein, bound chlorophyll b with a chlorophyll b content of approximately 4.4%. On the basis of these results, the evolution of pigment systems in an early stage of cyanobacterial development is discussed in this paper. (+info)
The protein disulfide isomerase-like RB60 is partitioned between stroma and thylakoids in Chlamydomonas reinhardtii chloroplasts.
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Translation of psbA mRNA in Chlamydomonas reinhardtii chloroplasts is regulated by a redox signal(s). RB60 is a member of a protein complex that binds with high affinity to the 5'-untranslated region of psbA mRNA. RB60 has been suggested to act as a redox-sensor subunit of the protein complex regulating translation of chloroplast psbA mRNA. Surprisingly, cloning of RB60 identified high homology to the endoplasmic reticulum-localized protein disulfide isomerase, including an endoplasmic reticulum-retention signal at its carboxyl terminus. Here we show, by in vitro import studies, that the recombinant RB60 is imported into isolated chloroplasts of C. reinhardtii and pea in a transit peptide-dependent manner. Subfractionation of C. reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids. The nature of association of native RB60, and imported recombinant RB60, with thylakoids is similar and suggests that RB60 is tightly bound to thylakoids. The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed. (+info)
Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii.
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To characterize genes whose expression is induced in carbon-stress conditions, 12,969 and 13,450 5'-end expressed sequence tags (ESTs) were generated from cells grown in low-CO2 and high-CO2 conditions of the unicellular green alga, Chlamydomonas reinhardtii. These ESTs were clustered into 4436 and 3566 non-redundant EST groups, respectively. Comparison of their sequences with those of 3433 non-redundant ESTs previously generated from the cells under the standard growth condition indicated that 2665 and 1879 EST groups occurred only in the low-CO2 and high-CO2 populations, respectively. It was also noted that 96.2% and 96.0% of the cDNA species respectively obtained from the low-CO2 and high-CO2 conditions had no similar EST sequence deposited in the public databases. The EST species identified only in the low-CO2 treated cells included genes previously reported to be expressed specifically in low-CO2 acclimatized cells, suggesting that the ESTs generated in this study will be a useful source for analysis of genes related to carbon-stress acclimatization. The sequence information and search results of each clone will appear at the web site: http://www.kazusa.or.jp/en/plant/chlamy/EST/. (+info)
RbcS suppressor mutations improve the thermal stability and CO2/O2 specificity of rbcL- mutant ribulose-1,5-bisphosphate carboxylase/oxygenase.
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In the green alga Chlamydomonas reinhardtii, a Leu(290)-to-Phe (L290F) substitution in the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco), which is coded by the chloroplast rbcL gene, was previously found to be suppressed by second-site Ala(222)-to-Thr and Val(262)-to-Leu substitutions. These substitutions complement the photosynthesis deficiency of the L290F mutant by restoring the decreased thermal stability, catalytic efficiency, and CO(2)/O(2) specificity of the mutant enzyme back to wild-type values. Because residues 222, 262, and 290 interact with the loop between beta strands A and B of the Rubisco small subunit, which is coded by RbcS1 and RbcS2 nuclear genes, it seemed possible that substitutions in this loop might also suppress L290F. A mutation in a nuclear gene, Rbc-1, was previously found to suppress the biochemical defects of the L290F enzyme at a posttranslational step, but the nature of this gene and its product remains unknown. In the present study, three nuclear-gene suppressors were found to be linked to each other but not to the Rbc-1 locus. DNA sequencing revealed that the RbcS2 genes of these suppressor strains have mutations that cause either Asn(54)-to-Ser or Ala(57)-to-Val substitutions in the small-subunit betaA/betaB loop. When present in otherwise wild-type cells, with or without the resident RbcS1 gene, the mutant small subunits improve the thermal stability of wild-type Rubisco. These results indicate that the betaA/betaB loop, which is unique to eukaryotic Rubisco, contributes to holoenzyme thermal stability, catalytic efficiency, and CO(2)/O(2) specificity. The small subunit may be a fruitful target for engineering improved Rubisco. (+info)
Amino acid residues that modulate the properties of tyrosine Y(Z) and the manganese cluster in the water oxidizing complex of photosystem II.
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The catalytic site for photosynthetic water oxidation is embedded in a protein matrix consisting of nearly 30 different polypeptides. Residues from several of these polypeptides modulate the properties of the tetrameric Mn cluster and the redox-active tyrosine residue, Y(Z), that are located at the catalytic site. However, most or all of the residues that interact directly with Y(Z) and the Mn cluster appear to be contributed by the D1 polypeptide. This review summarizes our knowledge of the environments of Y(Z) and the Mn cluster as obtained from the introduction of site-directed, deletion, and other mutations into the photosystem II polypeptides of the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii. (+info)