Large Subunit Of Ribulose Bisphosphate Carboxylase Research Articles

Purification and Properties of Ribulosebisphosphate Carboxylase Large Subunit Binding Protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

Differential Expression of the Ribulose Bisphosphate Carboxylase Large Subunit Gene in Bundle Sheath and Mesophyll Cells of Developing Maize Leaves Is Influenced by Light

We have investigated the influence of light on the mRNA of the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in mesophyll and bundle sheath cells of the C(4) plant Zea mays. The gene is transcribed in both cell types in leaves of seedlings grown in the dark. However, upon illumination, the level of mRNA declines and becomes undetectable in mesophyll cells after 72 hours. On the contrary, in bundle sheath cells the two transcripts of the same gene increase to peak after 24 hours of greening and then decrease to a steady state level. This study was made possible by the development of improved methods for the isolation of two maize leaf cell types suitable for the extraction of high quality RNAs at different developmental states, i.e. etiolated, greening and green.

DNA supercoiling affects in vitro transcription of two maize chloroplast genes differently.

Two adjacent, divergently transcribed, developmentally regulated genes of the maize chloroplast chromosome have different superhelical density/transcriptional activation profiles when transcribed in vitro by the homologous DNA-dependent RNA polymerase. Promoter-specific transcription of the gene for the beta and epsilon subunits of coupling factor 1 (cf1BE) increases and plateaus from templates of increasing negative superhelicity, while transcription of the gene for ribulose bisphosphate carboxylase large subunit (rcL) rises and then falls. Maximal transcription from the two promoters occurs at different template negative superhelical densities and transcription of the two genes is stimulated to different degrees. The different superhelicity profiles alter the molar ratios of the two transcripts over an order of magnitude. Changes in DNA conformation represent one possible mechanism for the differential regulation of the genes.

Inhibition and stimulation of ribulose-1,5-bisphosphate carboxylase/oxygenase by glyoxylate

Glyoxylate is a slowly reversible inhibitor of the CO 2 Mg 2+ -activated form of ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach leaves. Inactivation occurred with an apparent dissociation constant of 3.3 m mand a maximum pseudo-first-order rate constant of 7 × 10 −3 s −1. The rate constant for reactivation was 1.2 × 10 −2 s −1. Glyoxylate did not cause differential inhibition of ribulosebisphosphate carboxylase or oxygenase activities. 6-Phosphogluconate protected the enzyme from inactivation by glyoxylate. Glyoxylate was incorporated irreversibly into the large subunit of ribulosebisphosphate carboxylase after reduction with sodium borohydride. Activated enzyme incorporated 1.3 mol of glyoxylate per mole protomer, while enzyme treated with carboxyarabinitol 1,5-bisphosphate (CABP) to protect the active sites incorporated only 0.3 mol glyoxylate per mole protomer. The data suggest that glyoxylate forms a Schiff base with a lysyl residue in the region of the catalytic site. Glyoxylate stimulated the activity of the unactivated enzyme by about twofold. Pseudo-first-order inactivation also occurred with the unactivated enzyme after the initial stimulation by glyoxylate, although at a much slower rate than with the activated enzyme. Glyoxylate treatment of partially activated enzyme did not stimulate formation of the quaternary complex of enzyme · CO 2 · Mg 2+ · CABP.

In vivo chloroplast protein synthesis by the chromophytic alga Olisthodiscus luteus

Information on the ctDNA protein coding profile of the Chlorophyta, Rhodophyta, and Chromophyta might provide clues to the evolutionary mechanism(s) by which plants diverged into these three phylogenetic groups. The purpose of this study was to examine the ctDNA protein coding profile of the chromophytic plant Olisthodiscus luteus. Whole cells were labeled in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Control experiments demonstrate that the chloroplast proteins labeled in vivo by this technique form a distinct subset of the total proteins synthesized by the cell. Approximately 50 plastid proteins (35 soluble, 15 membrane) were detected after two-dimensional gel electrophoresis and fluorography. Three ctDNA-coded proteins, the large subunit of ribulosebisphosphate carboxylase, the apoprotein of the P700-chlorophyll a-protein complex, and the "photogene" were identified. These proteins are also coded by chlorophytic ctDNA. Unexpectedly, the ctDNA of Olisthodiscus was shown to code for the small subunit of ribulosebisphosphate carboxylase. The gene for this enzyme subunit is nuclear coded in all chlorophytic plants that have been analyzed.

Transcriptional activity of isolated maize chloroplasts

Chloroplasts and etioplasts, isolated from light- or dark-grown Zea mays plants, respectively, can incorporate labeled UTP into RNA in a reaction stimulated by light or ATP. This in organello RNA synthesis proceeded at a linear rate for up to 2 h. When expressed per unit protein, plastids from dark-grown plants incorporated more UTP than those from light-grown plants, and the highest rate of UTP incorporation was found in plastids from light-stimulated leaves (grown previously in the dark). The in organello newly synthesized RNA was heterodispersed, with most transcripts smaller than 14 S. Specific transcripts were detected in organelles from both darkand light-grown plants that contain sequences that are homologous to the mRNAs for the rbcL gene (coding for the large subunit of ribulose bisphosphate carboxylase (LS-RuBPCase)) and for the psbA gene (32-kDa thylakoid membrane protein). Qualitatively, the newly synthesized in organello transcripts were similar from the dark and light organelles.

Single gene for the large subunit of ribulosebisphosphate carboxylase in maize yields two differentially regulated mRNAs.

A second mRNA coding for the large subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] has been found in the plastids of maize leaves. The 5' terminus of this mRNA was shown by S1 nuclease analysis to be 238 nucleotides upstream of the previously described large subunit mRNA [McIntosh, L., Poulsen, C. & Bogorad, L. (1980) Nature (London) 288, 556-560]. The same two mRNAs were produced in a homologous in vitro transcription system using cloned plastid DNA as a template. The ratio of the two mRNAs changes during light-induced plastid development and can be altered in the in vitro system by manipulation of transcription conditions.

Extensive and widespread homologies between mitochondrial DNA and chloroplast DNA in plants

We used hybridization techniques to demonstrate that numerous sequence homologies exist between cloned mung bean and spinach chloroplast DNA (ctDNA) restriction fragments and mtDNAs from corn, mung bean, spinach, and pea. The strongest cross-homologies are between clones derived from the ctDNA inverted repeat and mtDNA from corn and pea, although all the ctDNA clones tested hybridized to at least one mtDNA restriction fragment. Known chloroplast genes showing strong mtDNA homologies include those for the large subunit of ribulosebisphosphate carboxylase, which hybridizes to corn mtDNA, and the beta subunit of the chloroplast ATPase, which hybridizes to mung bean mtDNA. Certain of these homologies were confirmed by using cloned spinach mtDNA restriction fragments as probes in reciprocal hybridizations to ctDNA. Several of these ctDNA-homologous mtDNA sequences were shown to be much more closely related to ctDNA from the same species than to that of a distantly related species. We interpret these differential homologies as evidence for relatively recent DNA sequence transfer events, suggesting that transpostion between the two genomes is an ongoing evolutionary process.

Amino-acid sequence of the large subunit of d-ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum

The complete amino acid sequence of the large subunit of the ribulosebisphosphate carboxylase (3-phospho- d-glycerate carboxy-lyase (dimirizing), ED 4.1.1. 39) from Nicotiana tabacum has been determined by alignment of tryptic, chymotryptic and cyanogen bromide fragments. The sequence is — except for positions 284 (glycine instead of cysteine) and 377 (valine instead of glutamic acid) — in agreement with the one deduced from gene sequencing (Shinozaki, K. and Sugiura, M. (1982) Gene 20, 91–102). However, the protein chemical determination yields additional information not evident from nucleotide sequencing: (1) The amino terminus is proteolytically processed and appears inhomogeneous; (2) The amino acid sequence is dimorphic in positions 394 and 405 (and possibly in position 23). The latter observation proves that for the large subunit of ribulosebisphosphate carboxylase from N. tabacum there exist at least two (if not more) slightly different genes.

Plastid translation in organello and in vitro during light-induced development in Euglena.

Plastids were isolated from dark-grown Euglena gracilis, from cells exposed to light for 3 h, and from normal, light-grown cells. Plastid protein synthesis was then measured either in organello, by incorporation of [35S]Met into isolated plastids, or in vitro, by programming wheat germ, reticulocyte, or Escherichia coli translation systems with RNA isolated from the plastids. Specific and total rates of protein synthesis in organello increased during light-induced development about 100-fold. In contrast, specific mRNA activity of plastid RNA increased no more than 3-fold, and the estimated total mRNA activity of plastids increased less than 10-fold. Protein synthesis in plastids appears therefore to be subject to strong regulation at a level other than that of transcription superimposed on weak transcriptional regulation. The synthesis of the large subunit of ribulose-bisphosphate carboxylase in particular appears to be under translational control. There is also qualitative evidence for regulation. Most plastid mRNAs, as indicated by translation in vitro, electrophoresis, and fluorography of their translation products, increase with light-induced development as a coordinate set. A second or proplastid set can be detected in RNAs from immature plastids, but not from mature chloroplasts. A few mRNAs appear only later in development, corresponding to a delayed set. Most translation products measured in organello also appear to belong to the coordinate set, a few to the proplastid and delayed sets, and a further few to a transient set, which appears only early in development.

Clone bank of Nicotiana tabacum chloroplast DNA: Mapping of the alpha, beta and epsilon subunits of the ATPase coupling factor, the large subunit of ribulosebisphosphate carboxylase, and the 32-kDal membrane protein

All of the Pst I restriction fragments of the chloroplast DNA of Nicotiana tabacum have been cloned in the plasmid vector pBR322. The cloned fragment sizes range from 0.8 to 26 kb, are stable, and can be amplified by chloramphenicol with varying efficiencies. Using these clones we have detailed a PstI physical map of the tobacco chloroplast genome. Selected clones of SalI, BamHl and PstI fragments were used to localize the map positions of the α, β, and ε subunits of the chloroplast ATPase coupling factor, the large subunit of ribulosediphosphate carboxylase and the 32-kDal membrane protein. The gene products of these clones were characterized by RNA transcript sizing, immunoprecipitation of maxicell-directed protein synthesis, and hybrid-arrested translation.

Protein changes in wheat infected with wheat streak mosaic virus and in barley infected with barley stripe mosaic virus

Proteins were isolated from third leaves of barley systemically infected with barley stripe mosaic virus, of wheat infected with wheat streak mosaic virus (WSMV) and of uninfected wheat and barley plants. Analysis of the proteins by sodium dodecyl sulphate gel electrophoresis showed that certain proteins were decreased and others increased in relative amount as a result of virus infection. The latter group of proteins, which were mostly different for each virus, included the viral coat proteins and may have included other virus-coded proteins. Many of the proteins present in decreased amounts were the same for the 2 viruses. One of these was identified as the large subunit of ribulosebisphosphate carboxylase, and others were tentatively identified as chloroplast associated. Decreases in concentrations of 4 polypeptides (two of molecular weight 52 000 and one each of 34 000 and 24 000) upon WSMV infection were confirmed by 2-dimensional gel analysis. Polypeptides of molecular weight 98 000–100 000, 72 000, 66 000, 43 000 and 29 000 were radioactively labelled in WSMV-infected leaves, but not in xminfected, in presence of chloramphenicol, alpha-amanitin, and actino-mycin D and presumably were virus coded. The protein of molecular weight 66 000 was present in higher concentration than the coat protein in infected leaves and was readily detected without labelling.

Sequence of the gene coding for the β-subunit of dinitrogenase from the blue-green alga Anabaena

The nitrogen fixation nif K gene of the blue-green alga Anabaena, which codes for the beta-subunit of dinitrogenase, has been subjected to sequence analysis. The nif K protein is predicted to be 512 amino acids long, to have a M(r) or 57,583, and to contain six cysteine residues. Three of these cysteines are within peptides homologous to FeS cluster-binding cysteinyl peptides from ferredoxins and from a high potential iron protein and, thus, may be ligands to which FeS clusters bind in dinitrogenase. The sequences surrounding the cysteine residues are 70% homologous to the corresponding cysteinyl tryptic peptides of the Azotobacter vinelandii dinitrogenase, although the positions of the cysteine residues are not always conserved between the two proteins. A 15-amino acid coding sequence precedes nif K on its transcript. Amino acid codon usage is highly asymmetric and parallels that found for the Anabaena dinitrogenase reductase gene (nif H). Putative promoter and ribosome binding site sequences were identified for nif K. These regulatory sequences are homologous to sequences preceding nif D; nif D codes for the alpha-subunit of dinitrogenase but is separated from nif K on the chromosome by 11,000 nucleotides. The nif K promoter also is virtually identical to a promoter-like sequence that immediately precedes the start of the transcript for the large subunit of ribulosebisphosphate carboxylase from maize chloroplasts. This homology appears to support the theory that chloroplasts evolved from blue-green algae.

Structures of the genes for the β and ε subunits of spinach chloroplast ATPase indicate a dicistronic mRNA and an overlapping translation stop/start signal

A 2,4-kilobase-pair region of spinach chloroplast DNA adjacent to the gene for the large subunit of ribulosebisphosphate carboxylase has been analyzed by RNA hybridization, in vitro transcription/translation, and DNA sequence determination. The analysis indicates that this region carries the genes for the beta and epsilon subunits of chloroplast ATPase and that the two genes are cotranscribed into a dicistronic mRNA with 4-base-pair overlap between the stop codon of the beta-subunit gene and the start codon of the epsilon-subunit gene. The ATPase and carboxylase genes are transcribed divergently with respect to each other. The deduced amino acid sequences of the beta and epsilon subunits from spinach show 67% and 26% homology, respectively, with the published sequences of the beta and epsilon subunits of Escherichia coli ATPase.

Amplification of maize ribulose bisphosphate carboxylase large subunit synthesis in E. coli by transcriptional fusion with the lambda N operon.

The maize chloroplast gene coding for the large subunit of ribulose bisphosphate carboxylase (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) has been placed under the transcriptional control of the bacteriophage lambda promoter PL, by fusion with the lambda N operon located on a multicopy plasmid. Transcription from PL was repressed at 32 degrees C by the presence in the E. coli chromosome of a cIts gene that specifies a temperature-sensitive repressor. After inactivation of the repressor at 45 degrees C unmoderated transcription of the chloroplast gene occurred from the PL promoter. Translation was probably initiated from a chloroplast Shine-Dalgarno sequence located five nucleotides from the N-terminal methionine initiation codon to yield a polypeptide the same size as that synthesised in maize. This direct translation results in a level of expression of the chloroplast gene corresponding to approximately 2% of the total E. coli cell protein as ribulose bisphosphate carboxylase large subunits. Transcriptional fusions with the lambda N operon should provide a generally applicable, simple method for the amplification and regulation of chloroplast gene expression in E. coli.

Differential transcription in vivo and in vitro of two adjacent maize chloroplast genes: The large subunit of ribulosebisphosphate carboxylase and the 2.2-kilobase gene

The transcription of cloned maize plastid DNA sequences in vitro by maize plastid DNA-dependent RNA polymerase has been studied to expose the roles of the enzyme, polypeptide cofactors, and DNA sequences in the regulation of gene expression. The 4.35-kilobase pair BamHI fragment 9 carries the maize plastid gene for the large subunit of ribulosebisphosphate carboxylase and part of the gene for a 2.2-kilobase RNA. These two genes are separated by approximately 330 base pairs and are transcribed divergently. Transcripts of the gene for the large subunit of ribulosebisphosphate carboxylase are abundant in bundle sheath cells of maize leaves and we show here that transcripts of the 2.2-kilobase RNA gene are present in both mesophyll cells and the adjacent bundle sheath cells. In vitro, in the presence of the S factor, maize chloroplast DNA-dependent RNA polymerase produces a transcript of the gene for the large subunit of ribulose-bisphosphate carboxylase with a 5' terminus like that of the corresponding mRNA isolated from plastids, transcribes chloroplast DNA sequences of Bam fragment 9 in a chimeric plasmid in preference to the vehicle RSF 1030 and, in a ratio of 3:1, preferentially transcribes the gene for the large subunit of ribulosebisphosphate carboxylase over the 2.2-kilobase RNA gene from supercoiled chimeric plasmid DNA.

Glyphosine, a plant growth regulator, affects chloroplast membrane proteins

Glyphosine ( N, N-bis(phosphonomethyl)glycine) is known to increase sucrose levels in sugarcane and to cause chlorosis in maize and other plants. It has been suggested (Crofts, S.M., Arntzen, C.J., Vanderhoef, L.N. and Zettinger, C.S. (1974) Biochim. Biophys. Acta 335, 211–217) that its primary mode of action is to inhibit the synthesis of plastid rRNA. Growth of Lemna gibba L. G-3 on 5 · 10 −4M glyphosine causes the plants to produce fronds lacking chlorophyll. The plastids in these white fronds contain only a few internal membrane structures, some of which are stacked. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows an accumulation of substantial amounts of both the large and small subunits of ribulosebisphosphate carboxylase by the white fronds. The membrane fraction from these fronds contains only traces of the light-harvesting chlorophyll a b apoprotein in comparison to control plants. In vivo labeling and immunoprecipitation show that the large subunit of ribulose-bisphosphate carboxylase is actively synthesized by the white fronds. However, labeling of the chlorophyll a b apoprotein and a 32000 dalton protein in the membrane fraction is extremely low compared to control plants. We conclude that in Lemna, glyphosine differentially affects the synthesis and/or processing of soluble proteins and some membrane chloroplast proteins, and could be useful in understanding the biogenesis of chloroplast membranes.

Cloning and mapping of the chloroplast DNA sequences for two messenger RNAs from mustard (Sinapis alba L.).

Restriction fragments of chloroplast (cp)DNA from mustard leaves were cloned in E. coli using pBR 322 as the vehicle. Cloned fragments containing the structural sequences for two polypeptides of 56,000 and 35,000 daltons were selected by a mRNA hybridization-translation procedure. The cloned mustard genes are structurally related to chloroplast genes from maize. They hybridize to the maize mRNAs for the large subunit of ribulosebisphosphate carboxylase and for the 34,500 dalton precursor to a membrane protein. The coding sequence on mustard cpDNA for the 56,000 dalton polypeptide is colinear with a 1,500 base pair transcript, and the sequence for the 35,000 dalton polypeptide is colinear with a 1,220 base pair transcript.

Chloroplast gene sequence for the large subunit of ribulose bisphosphatecarboxylase of maize

The chloroplast gene coding for the large subunit of ribulose 1,5-bisphosphatecarboxylase in Zea mays has been sequenced. It contains neither a ‘classical’ prokaryotic promoter sequence nor its proposed eukaryotic counterpart but does have a typical prokaryotic ribosome binding site close to the site at which translation is initiated. Almost all possible codons occur and are translated by the ‘universal code’ rather than by the variation of it found in mitochondria.

Peptide mapping of the ribulose bisphosphate carboxylase small subunit from the somatic hybrid of tomato and potato

Five somatic hybrids of potato and tomato obtained by protoplast fusion were identified using the isoelectric focusing patterns of ribulose bisphosphate carboxylase as phenotypic markers of the nuclear and chloroplast genomes. Together with the previously analyzed hybrids (14) this brings the number of verified somatic hybrids between the two species to nine. The products of both tomato and potato nuclear genomes are present in the ribulose bisphosphate carboxylase oligomer from all nine hybrids. Four of the hybrids contain the tomato large subunit and hence the tomato chloroplast DNA is functional, whereas in five hybrids the potato ribulose bisphosphate carboxylase large subunit and thereby the potato chloroplast DNA is present. The former are designated topatoes and the latter pomatoes (11).Mapping of chymotryptic peptides of the small subunit of tomato and potato revealed a tyrosine containing peptide with species specific mobility. Peptide maps from an analyzed somatic hybrid contained both the tyrosine peptide specific for potato and that specific for tomato. Maps of tryptic peptides of the small subunit of tomato differed from those of the potato in 2 peptides. The tryptic peptide maps of one analyzed hybrid contained the two peptides unique to potato as well as the two peptides unique to tomato. The tomato-potato hybrids are aneuploid with chromosome numbers ranging from 48 to 74, the expected allotetraploid number being 48 and the expected allohexaploid number 72.