Large Subunit Of Ribulose Bisphosphate Research Articles

DNA Analysis of Natural Fiber Rope*

When rope is found at a crime scene, the type of fiber is currently identified through its microscopic characteristics. However, these characteristics may not always unambiguously distinguish some types of rope from others. If rope samples contain cells from the plants of origin, then DNA analysis may prove to be a better way to identify the type of rope obtained from a crime scene. The objective of this project was to develop techniques of DNA analysis that can be used to differentiate between ropes made from Cannabis sativa L. (hemp), Agave sisalana Perrine (sisal), Musa textilis Née (abaca, "Manila hemp"), Linum usitatissimum L. (flax), and Corchorus olitorus L. (jute). The procedures included extracting the DNA from the rope, performing polymerase chain reaction (PCR) using the extracted DNA as a template, and analyzing the DNA products. A primer pair for PCR, chosen from within a chloroplast gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, was designed to be specific for plant DNA and complementary to the genes from all five plants. The resulting PCR fragments were approximately 771 base pairs long. The PCR fragments, distinguished through base sequence analysis or restriction enzyme analysis, could be used to identify the five different rope types. The procedure provides a useful addition to visual methods of comparing rope samples.

Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.

The rbcL genes of two Cuscuta species, C. gronovii and C. subinclusa, are transcribed by the nuclear-encoded plastid RNA polymerase (NEP).

Some species of the holoparasitic flowering plant genus Cuscuta, like C. reflexa, have retained a plastid genome that encodes photosynthesis-related gene products as well as the plastid-encoded RNA polymerase (PEP). In contrast, other species like C. gronovii and C. subinclusa have lost the rpo genes coding for the PEP subunits while photosynthetic genes have been retained. In order to ensure expression of the photosynthesis-related genes in the absence of PEP, a number of adaptations within the plastid genome were required that enable gene transcription mediated exclusively by the nuclear-encoded plastid RNA polymerase (NEP). In this study we analyzed promoter sequence conservation and transcription start sites of a typical PEP gene of non-parasitic plants, rbcL, which codes for the large subunit of ribulose bisphosphate carboxylase/oxygenase. We show that despite high sequence conservation of the coding region of rbcL among different Cuscuta species and tobacco, the 5' non-coding regions of C. gronovii and C. subinclusa have suffered extensive deletions encompassing the PEP promoter that is present in C. reflexa and tobacco. Primer-extension analyses enabled the identification of transcripts initiated at NEP promoter motifs in C. gronovii and C. subinclusa that are not detectable in the 5' non-coding region of C. reflexa.

A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO.

The genomes of several nonphotosynthetic bacteria, such as Bacillus subtilis, and some Archaea include genes for proteins with sequence homology to the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO). We found that such a RuBisCO-like protein (RLP) from B. subtilis catalyzed the 2,3-diketo-5-methylthiopentyl-1-phosphate enolase reaction in the methionine salvage pathway. A growth-defective mutant, in which the gene for this RLP had been disrupted, was rescued by the gene for RuBisCOfrom the photosynthetic bacterium Rhodospirillum rubrum. Thus, the photosynthetic RuBisCO from R. rubrum retains the ability to function in the methionine salvage pathway in B. subtilis.

Diel rhythms in ribulose-1,5-bisphosphate carboxylase/oxygenase and glutamine synthetase gene expression in a natural population of marine picoplanktonic cyanobacteria (Synechococcus spp.).

Diel periodicity in the expression of key genes involved in carbon and nitrogen assimilation in marine Synechococcus spp. was investigated in a natural population growing in the surface waters of a cyclonic eddy in the northeast Atlantic Ocean. Synechococcus sp. cell concentrations within the upper mixed layer showed a net increase of three- to fourfold during the course of the experiment (13 to 22 July 1991), the population undergoing approximately one synchronous division per day. Consistent with the observed temporal pattern of phycoerythrin (CpeBA) biosynthesis, comparatively little variation was found in cpeBA mRNA abundance during either of the diel cycles investigated. In marked contrast, the relative abundance of transcripts originating from the genes encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) and glutamine synthetase (glnA) showed considerable systematic temporal variation and oscillated during the course of each diel cycle in a reciprocal rhythm. Whereas activation of rbcL transcription was clearly not light dependent, expression of glnA appeared sensitive to endogenous changes in the physiological demands for nitrogen that arise as a natural consequence of temporal periodicity in photosynthetic carbon assimilation. The data presented support the hypothesis that a degree of temporal separation may exist between the most active periods of carbon and nitrogen assimilation in natural populations of marine Synecoccoccus spp.

Effect of mutation of lysine-128 of the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans.

The contribution of lysine-128 within the active site of Anacystis nidulans d-ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was investigated by the characterization of mutants in which lysine-128 was replaced with arginine, glycine, glutamine, histidine or aspartic acid. Mutated genes encoding the Rubisco large subunit were expressed in Escherichia coli and the resultant polypeptides assembled into active complexes. All of the mutant enzymes had a lower affinity for ribulose 1,5-bisphosphate (RuBP) and lower rates of carboxylation. Substitution of lysine-128 with glutamine, histidine or aspartic acid decreased the specificity factor and led to the production of an additional monophosphate reaction product. We show that this product results from the loss of the phosphate from C-1 of RuBP, most probably by beta-elimination from the 2,3-enediolate derivative of RuBP. The results confirm that lysine-128 is important in determining the position of the essential epsilon-amino group of lysine-334 within the active site and in loop dynamics. This further demonstrates that residues remote from the active site can be manipulated to modify catalytic function.

A study of conserved in-loop and out-of-loop glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase by directed mutagenesis.

The replacement of all 22 completely conserved glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans by directed mutagenesis is described. In each beta/alpha barrel of the large subunit there are 12 completely conserved glycines in six of eight loops at the C-termini of eight beta-strands and four in loops at N-terminal ends of the beta-strands. Two completely conserved glycines are also in each beta/alpha barrel backbone and four more are in a large N-terminal portion preceding the barrel in a given L subunit. Substitution of glycines in loops that are C-terminal to beta-strands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to catalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops. In contrast, substitution of glycines at the N-terminal ends of beta-strands in the beta/alpha barrel more often led to failure to detect L subunits or their assembly into L8S8 complex. Substitution of these and the other conserved glycines by proline was more deleterious to carboxylase activity than by alanine in enzymes that assembled.

Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning.

Amino acids composing a flexible loop (loop 6) of the eight-stranded barrel domain of the L-subunit of Synechococcus ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) involved in reaction intermediate stabilization have been modified by site-specific mutagenesis. Changes at positions both distant and within the active site affect overall catalysis and substrate partitioning. Most significantly, replacement of the active site Lys (Lys-334) with Arg at the apex of the loop almost completely suppressed the carboxylase activity of the enzyme relative to oxygenation, with only a modest reduction in overall catalysis. Val-331 and Thr-342, more distant from the active site but with interacting side chains, were changed to larger and smaller residues with differential effects on both turnover and substrate partitioning. Substitution of the loop with the sequence found in more efficient carboxylases only increased partitioning marginally when accompanied by alterations in the C-terminal tail of the L-subunit that interacts with the loop. Generally, modifications to the loop composition also affected enediol formation, the first step of catalysis, suggesting that the geometry and hence flexibility of this segment affect more than just stabilization of the intermediates immediately following reaction with CO2 or O2.

Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination.

The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3'-truncated gene copy into the psb A-I locus. Both rbcL genes, in the psb A-I region and at the rbc locus, were non-functional; The former due to the 3' truncation, and the latter due to a deletion in the 5'-region (creating a 5' truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO2-requiring mutant ('cyanorubrum'). The 3' and the 5' truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L8S8) and R. rubrum (L2) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.

Protection of Tryptic-Sensitive Sites in the Large Subunit of Ribulosebisphosphate Carboxylase/Oxygenase by Catalysis

Limited tryptic proteolysis of spinach (Spinacia oleracea) ribulose bisphosphate carboxylase/oxygenase (ribulose-P(2) carboxylase) resulted in the ordered release of two adjacent N-terminal peptides from the large subunit, and an irreversible, partial inactivation of catalysis. The two peptides were identified as the N-terminal tryptic peptide (acetylated Pro-3 to Lys-8) and the penultimate tryptic peptide (Ala-9 to Lys-14). Kinetic comparison of hydrolysis at Lys-8 and Lys-14, enzyme inactivation, and changes in the molecular weight of the large subunit, indicated that proteolysis at Lys-14 correlated with inactivation, while proteolysis at Lys-8 occurred much more rapidly. Thus, enzyme inactivation is primarily the result of proteolysis at Lys-14. Proteolysis of ribulose-P(2) carboxylase under catalytic conditions (in the presence of CO(2), Mg(2+), and ribulose-P(2)) also resulted in ordered release of these tryptic peptides; however, the rate of proteolysis at lysyl residues 8 and 14 was reduced to approximately one-third of the rate of proteolysis of these lysyl residues under noncatalytic conditions (in the presence of CO(2) and Mg(2+) only). The protection of these lysyl residues from proteolysis under catalytic conditions could reflect conformational changes in the N-terminal domain of the large subunit which occur during the catalytic cycle.

Protein Methylation in Pea Chloroplasts

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [(3)H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. One is a polypeptide with a molecular mass of 64 kD, a second has an M(r) of 48 kD, and the third has a molecular mass of less than 10 kD. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide, having a molecular mass of 24 kD, is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methyl-linkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [(3)H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [(3)H]methyl group.

A critical arginine in the large subunit of ribulose bisphosphate carboxylase/oxygenase identified by site-directed mutagenesis.

Rapid inactivation by phenylglyoxal of ribulose bisphosphate carboxylase/oxygenase (ribulose-P2 carboxylase) from the cyanobacterium Anacystis nidulans suggests the presence of an essential arginine, the modification of which is reduced in the presence of the substrate ribulose bisphosphate. Arginine 292 in the large subunit of ribulose-P2 carboxylase from A. nidulans was chosen for site-directed mutagenesis studies on the basis of the complete conservation of this residue in corresponding sequences of ribulose-P2 carboxylase from divergent organisms. Arginine 292 was changed to leucine and to lysine by directed mutagenesis using suitable plasmids and the bacteriophage M13. Both substitutions resulted in the production of purifiable holoenzyme with no activity after expression in Escherichia coli.

Chloroplast DNA in Pinus monticola

Restriction sites on the chloroplast genome of Pinus monticola have been mapped, and the gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase, the genes for the photosystem II polypeptides psbA, psbD and psbC, and the 16S and 23S ribosomal RNA genes have been located. The genome lacks the large inverted repeat characteristic of most angiosperms. The gene order is similar to that found in P. radiata. The presence of dispersed repeated sequences is likely. Two structural features, lack of a large inverted repeat and the presence of dispersed repeats, may confer a degree of variability on the genome which will prove useful in studies of population structure.

Changes in polypeptide composition of Synechocystis sp. strain 6308 phycobilisomes induced by nitrogen starvation

Phycobilisomes isolated from actively growing Synechocystis sp. strain 6308 (ATCC 27150) consist of 12 polypeptides ranging in molecular mass from 11.5 to 95 kilodaltons. The phycobilisome anchor and linker polypeptides are glycosylated. Nitrogen starvation causes the progressive loss of phycocyanin and allophycocyanin subunits with molecular masses between 16 and 20 kilodaltons and of two linker polypeptides with molecular masses of 27 and 33 kilodaltons. Nitrogen starvation also leads to enrichment of four additional polypeptides with molecular masses of 46, 53, 57, and 61 kilodaltons and a transient enrichment of 35- and 41-kilodalton polypeptides in isolated phycobilisomes. The 57-kilodalton additional polypeptide was identified by immunoblotting as the large subunit of ribulosebisphosphate carboxylase/oxygenase. Proteins with the same molecular weights as the additional polypeptides were also coisolated with the 12 phycobilisome polypeptides in the supernatant of nitrogen-replete Synechocystis thylakoid membranes extracted in high-ionic-strength buffer and washed with deionized water. These observations suggest that the additional polypeptides in phycobilisomes from nitrogen-starved cells may be soluble or loosely bound membrane proteins which associate with phycobilisomes. The composition and degree of association of phycobilisomes with soluble and adjacent membrane polypeptides appear to be highly dynamic and specifically regulated by nitrogen availability. Possible mechanisms for variation in the strength of association between phycobilisomes and other polypeptides are suggested.

Post-translational modifications in the large subunit of ribulose bisphosphate carboxylase/oxygenase.

Two adjacent N-terminal tryptic peptides of the large subunit of ribulose bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] from spinach, wheat, tobacco, and muskmelon were removed by limited tryptic proteolysis. Characterization by peptide sequencing, amino acid composition, and tandem mass spectrometry revealed that the N-terminal residue from the large subunit of the enzyme from each plant species was acetylated proline. The sequence of the penultimate N-terminal tryptic peptide from the large subunit of the spinach and wheat enzyme was consistent with previous primary structure determinations. However, the penultimate N-terminal peptide from the large subunit of both the tobacco and muskmelon enzymes, while identical, differed from the corresponding peptide from spinach and wheat by containing a trimethyllysyl residue at position 14. Thus, tryptic proteolysis occurred at lysine-18 rather than lysine-14 as with the spinach and wheat enzymes. A comparison of the DNA sequences for the large subunit of ribulose bisphosphate carboxylase/oxygenase indicates that the N terminus has been post-translationally processed by removal of methionine-1 and serine-2 followed by acetylation of proline-3. In addition, for the enzyme from tobacco and muskmelon a third post-translational modification occurs at lysine-14 in the form of N epsilon-trimethylation.

Localization of multiple TMV encapsidation initiation sites on rbcL gene transcripts

TMV capsid protein reacts with and encapsidates many of the chloroplast DNA transcripts both in vivo and in vitro to form pseudovirions. We report on the encapsidation initiation reaction with one of the major RNA species found in in vivo formed pseudovirions, the mRNA for the chloroplast-encoded large subunit of ribulose bisphosphate carboxylase/oxygenase ( rbcL). This mRNA is found to contain at least three sites which are independently capable of reacting with capsid protein oligomers to initiate encapsidation. All three sites react with capsid protein less efficiently in vitro than does the functional viral RNA encapsidation initiation site (ei). The 5′ portion of the region that contains the most reactive rbcL site, ei-3, shows significant nucleotide sequence homology with the encapsidation initiation sites of the U1 and Cc strains of TMV and it can assume a folding structure that resembles that postulated for the Cc strain site. A site that acts as a block to rod elongation is present in transcripts from the region just 3′ to the segment from which the rbcL mRNA is transcribed, probably close to, or at the transcription termination signal.

A Five‐Base‐Pair‐Deletion in the Gene for the Large Subunit Causes the Lesion in the Ribulose Bisphosphate Carboxylase/Oxygenase‐Deficient Plastome Mutant Sigma of Oenothera hookeri

AbstractThe gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL) has been mapped on the Oenothera hookeri plastid chromosome. It is located close to the gene for the herbicide‐binding “32 kd” protein of the photosystem II reaction center (psbA), at a position different from that found in the ancestral angiosperm type of plastid chromosomes, due to an inversion in the large single‐copy region. The gene codes for a polypeptide of 475 amino acid residues corresponding to a molecular mass of 52.7 kd. The deduced amino acid composition diverges by 4.8% from the amino acid sequence of the spinach protein and by 8.2% from that of maize. The corresponding nucleotide sequences differ by 8.5 % and 15 % from each other. The rbcL gene of the RuBPcase/oase‐deficient Oenothera plastome mutant sigma contains a TTAAC deletion at amino acid residues 270/271 which introduces a frame shift and an amber stop codon seven triplets later. This lesion which probably arose by slipped mispairing is consistent with the previously observed, virtually full‐length mRNA that is decoded into a truncated large subunit polypeptide of approximately 30 kd in vitro and in vivo.

Variability of 3 cytoplasmically encoded proteins in the Triticum genus

The comparison of two-dimensional protein patterns from wheat alloplasmic lines (etiolated seedling stage) has allowed us to define three variable polypeptides with a cytoplasmic determination: the large subunit of ribulose bisphosphate carboxylase/oxygenase and two other polypeptides that are likely to be two forms of the chloroplastic beta subunit of ATP synthase. Two cytoplasmic types are found among the Triticum species. Of the diploids studied only T. speltoides and T. aucheri had these same cytoplasmic markers as T. aestivum. T. turgidum and T. timopheevi.

Daffodil Chromoplast DNA: Comparison with Chloroplast DNA, Physical Map, and Gene Localization

Abstract In daffodil, development of chloroplasts or chromoplasts is not accompanied by plastid DNA modification. This has been shown by comparing the fragment pattern of different restriction endonucleases, and by the lack of methylation of CCGG sequences. A physical map has been constructed for the plastome using the restriction endonucleases Sal I, Pst I, and Bgl I. The fragments containing the genes for the large subunit of ribulose bisphosphate carboxylase/oxygenase (rbcL), the alpha, beta, and epsilon subunits of the ATP synthase complex (atpA , atpB, atpE ), cytochrome f(petA ), and for the 51 kDa chlorophyll apoprotein of photosystem II (psbB) have been identified. The respective gene locations correspond to those on spinach chloroplast DNA.

An efficient and prolonged [formula omitted] translational system from isolated cucumber etioplasts

Etioplasts were isolated from dark grown cucumber cotyledons pretreated with kinetin and gibberellic acid. When incubated in a cofactor enriched medium these etioplasts incorporated [ 35S] methionine into a hot trichloroacetic acid-insoluble fraction; this incorporation was linear for 8 h of incubation and was inhibited by chloramphenicol but not by cycloheximide. Over the same time period, the etioplasts showed continued linear synthesis of the chlorophyll precursor protochlorophyllide, Mg-protoporphyrin and protoporphyrin IX. Analysis of products of in vitro protein synthesis by etioplasts and cotyledons showed the thylakoid membrane polypeptide profiles to be identical. Continued incorporation of [ 35S] methionine into the large subunit of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) for 8 h has been confirmed further by immuno-precipitation with anti-spinach RubisCO. This competent in vitro translation system should be useful for future studies of chloroplast protein synthesis and gene expression.