Rye photosystem II. Nucleotide sequence of the psbC gene coding 43- kDa chlorophyll(a)-binding proteins

The structure of the rye chloroplast DNA, which contains psbC gene coding for 43-kDa chlorophyll(a)-binding subunit of photosystem II, is determined. The sequence of trnS (UGA) gene encoding tRNA Ser is located at a distance of 140 bp downstream from the stop codon of psbC gene on the opposite DNA strand. The 5'-terminal part of psbC gene, like in other plants, overlaps by 50 bp the 3'-terminal region of psbD gene coding for D2 protein of photosystem II. The amino acid sequence of the psbC gene product reveals common features with the structure of the psbB gene product (CPa-1 protein). The structural similarity of these two proteins seems to reflect their similar functions.     

Three-dimensional model and characterization of the iron stress-induced CP43'-photosystem I supercomplex isolated from the cyanobacterium Synechocystis PCC 6803

The cyanobacterium Synechocystis PCC 6803 has been subjected to growth under iron-deficient conditions. As a consequence, the isiA gene is expressed, and its product, the chlorophyll a-binding protein CP43', accumulates in the cell. Recently, we have shown for the first time that 18 copies of this photosystem II (PSII)-like chlorophyll a-binding protein forms a ring around the trimeric photosystem I (PSI) reaction center (Bibby, T. S., Nield, J., and Barber, J. (2001) Nature, 412, 743-745). Here we further characterize the biochemical and structural properties of this novel CP43'-PSI supercomplex confirming that it is a functional unit of approximately 1900 kDa where the antenna size of PSI is increased by 70% or more. Using electron microscopy and single particle analysis, we have constructed a preliminary three-dimensional model of the CP43'-PSI supercomplex and used it as a framework to incorporate higher resolution structures of PSI and CP43 recently derived from x-ray crystallography. Not only does this work emphasize the flexibility of cyanobacterial light-harvesting systems in response to the lowering of phycobilisome and PSI levels under iron-deficient conditions, but it also has implications for understanding the organization of the related chlorophyll a/b-binding Pcb proteins of oxychlorobacteria, formerly known as prochlorophytes.     

Targeted interruption of the psaA and psaB genes encoding the reaction-centre proteins of photosystem I in the filamentous cyanobacterium Anabaena variabilis ATCC 29413

The two reaction-centre proteins of the photosystem I (PSI) complex are encoded by two adjacent genes named psaA and psaB. We have performed targeted mutagenesis to insertionally inactivate each of these genes in the filamentous cyanobacterium Anabaena variabilis ATCC 29413. The resulting mutant strains, termed psaA:: Nm^R and psaB:: Nm^R, were blue because of a high ratio of phycobilin to chlorophyll and were unable to grow in light. These mutant cells also lacked chemically reducible P700 (the reaction-centre chlorophylls of PSI) and as a consequence did not exhibit any PSI-mediated photochemical activity. However, their photosystem II (PSII) complexes were fully active. The loss of the PsaA and PsaB proteins and their associated chlorophyll molecules resulted in a five- to sevenfold decrease in the chlorophyll/PSII ratio in the mutant cells relative to the wild-type cells. Interestingly, the psaS:: Nm^R and not the psaA:: Nm^R mutant strain retained a small fluorescence peak (77K) at 721 nm originating from chlorophyll molecule(s) presumably bound to a small amount of the PsaA protein present in the psaB mutant. These results demonstrate that this organism is suitable for the manipulation of PSI reaction-centre proteins.     

Anti-sense RNA efficiently inhibits formation of the 10 kd polypeptide of photosystem II in transgenic potato plants: analysis of the role of the 10 kd protein.

A chimeric gene encoding an anti-sense RNA of the 10 kd protein of the water-splitting apparatus of photosystem II of higher plants under the control of the CaMV 35S promoter was introduced into potato using Agrobacterium based vectors. The expression of the anti-sense RNA led to a significant reduction of the amounts of the 10 kd protein and RNA in a number of transgenic plants. In three out of 36 plants tested, the level of the 10 kd protein was only up to 1-3% compared with the wild-type control. The drastic reduction of the 10 kd protein did not influence the accumulation of other photosystem II associated polypeptides at both the RNA and protein level. Furthermore no phenotypic differences were observed between potato plants expressing wild-type and drastically reduced levels of the 10 kd protein with respect to growth rate, habitus or ultrastructure of the chloroplasts. Measurements of the relaxation of the flash-induced enhancement in the fluorescence quantum yield as determined in intact leaves and the rates and characteristic oscillation pattern of O2 evolution as determined in isolated thylakoid samples however, show that the elimination of the 10 kd protein on the one hand retards reoxidation of QA- and on the other hand introduces a general disorder into the PSII complex.     

Isolation of cDNA clones for fourteen nuclear-encoded thylakoid membrane proteins

Summary Spinach cDNA libraries, made from polyadenylated seedling RNA, have been constructed in pBR322 and the expression vector gt11. Recombinant plasmids or phage for 14 intrinsic and peripheral thylakoid membrane proteins and one stromal protein have been identified. They encode components containing antigenic determinants against the lysine-rich 34 kd, the 23 kd and 16 kd proteins all associated with the water-splitting apparatus of the photosystem II reaction center, the ATP synthase subunits gamma, delta and CF_o-II, the Rieske Fe/S protein of the cytochrome b/f complex, subunits 2, 3, 5 and 6 of the photosystem I reaction center, plastocyanin, ferredoxin oxidoreductase, chlorophyll a/b-binding apoproteins of the lightharvesting complex associated with photosystem II, and the small subunit of the stromal enzyme ribulose bisphosphate corboxylase/oxygenase. The cDNA inserts lack complementarity to plastid DNA but hybridize to restricted nuclear DNA as well as to discrete poly A⁺-mRNA species. The precursor products obtained after translation of hybrid selected RNA fractions in a wheat germ assay are imported and processed by isolated unbroken spinach chloroplasts. The imported components comigrate with the respective authentic proteins.     

Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942

The genome of the cyanobacterium Synechococcus sp. PCC7942 contains two genes encoding the D2 polypeptide of photosystem II (PSII), which are designated here as psbDI and psbDII. The psbDI gene, like the psbD gene of plant chloroplasts, is cotranscribed with and overlaps the open reading frame of the psbC gene, encoding the PSII protein CP43. The psbDII gene is not linked to psbC, and appears to be transcribed as a monocistronic message. The two psbD genes encode identical polypeptides of 352 amino acids, which are 86% conserved with the D2 polypeptide of spinach. In plants, the translational start codon of the psbC gene has been reported to be an ATG codon 50 bp upstream from the end of the psbD gene. This triplet is not present in the psbDI sequence of Synechococcus sp., but is replaced by ACG, a codon which is very unlikely to initiate translation. Translation of the psbC gene may begin at a GTG codon which overlap the psbDI open reading frame by 14 bp and is preceded by a block of homology to the 3' end of the 16S ribosomal RNA, a potential ribosome-binding site. There are only two bp differences between the sequences of the two psbD genes; one of these results in substitution in psbDII of GCG for the presumed GTG start codon in psbDI.     

Mytilus mitochondrial DNA contains a functional gene for a tRNASer(UCN) with a dihydrouridine arm-replacement loop and a pseudo-tRNASer(UCN) gene.

A 2500-nucleotide pair (ntp) sequence of F-type mitochondrial (mt) DNA of the Pacific Rim mussel Mytilus californianus (class Bivalvia, phylum Mollusca) that contains two complete (ND2 and ND3) and two partial (COI and COIII) protein genes and nine tRNA genes is presented. Seven of the encoded tRNAs (Ala, Arg, His, Met(AUA), Pro, Ser(UCN), and Trp) have the potential to fold into the orthodox four-armed tRNA secondary structure, while two [tRNASer(AGN) and a second tRNASer(UCN)] will fold only into tRNAs with a dihydrouridine (DHU) arm-replacement loop. Comparison of these mt-tRNA gene sequences with previously published, corresponding M. edulis F-type mtDNA indicates that similarity between the four-armed tRNASer(UCN) genes is only 63.8% compared with an average of 92.1% (range 86.2-98. 5%) for the remaining eight tRNA genes. Northern blot analysis indicated that mature tRNAs encoded by the DHU arm-replacement loop-containing tRNASer(UCN), tRNASer(AGN), tRNAMet(AUA), tRNATrp, and tRNAPro genes occur in M. californianus mitochondria, strengthening the view that all of these genes are functional. However, Northern blot and 5' RACE (rapid amplification of cDNA ends) analyses indicated that the four-armed tRNASer(UCN) gene is transcribed into a stable RNA that includes the downstream COI sequence and is not processed into a mature tRNA. On the basis of these observations the M. californianus and M. edulis four-armed tRNASer(UCN) sequences are interpreted as pseudo-tRNASer(UCN) genes.     

Nuclear and chloroplast mutations affect the synthesis or stability of the chloroplast psbC gene product in Chlamydomonas reinhardtii.

The psbC gene of Chlamydomonas reinhardtii encodes P6, the 43 kd photosystem II core polypeptide. The sequence of P6 is highly homologous to the corresponding protein in higher plants with the exception of the N-terminal region where the first 12 amino acids are missing. Translation of P6 is initiated at GUG in C. reinhardtii. The chloroplast mutant MA16 produces a highly unstable P6 protein. The mutation in this strain maps near the middle of the psbC gene and consists of a 6 bp duplication that creates a Ser-Leu repeat at the end of one transmembrane domain. Two nuclear mutants, F34 and F64, and one chloroplast mutant, FuD34, are unable to synthesize P6. All of these mutants accumulate wild-type levels of psbC mRNA. The FuD34 mutation has been localized near the middle of the 550 bp 5' untranslated region of psbC where the RNA can be folded into a stem-loop structure. A chloroplast suppressor of F34 has been isolated that partially restores synthesis of the 43 kd protein. The mutation of this suppressor is near that of FuD34, in the same stem-loop region. These chloroplast mutations appear to define the target site of a nuclear factor that is involved in P6 translation.     

Sequence of two genes in pea chloroplast DNA coding for 84 and 82 kD polypeptides of the photosystem I complex

Summary The genes encoding the two P700 chlorophyll a-apoproteins of the photosystem I complex were localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the genes and the flanking regions has been determined. The genes are separated by 25 bp and are probably cotranscribed. The 5 terminal gene (psaA1) codes for a 761-residue protein (MW 84.1 kD) and the 3 terminal gene (psaA2) for a 734-residue protein (MW 82.4 kD). Both proteins are highly hydrophobic and contain eleven putative membrane-spanning domains. The homology to the corresponding polypeptides from maize are 89% and 95% for psaA1 and psaA2, respectively. A putative promoter has been identified for the psaA1 gene, and potential ribosome binding sites are present before both genes.