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At least 19 recordsLinked to original sources
PubMed2000

The unicellular green alga Chlamydomonas reinhardtii as an experimental system to study chloroplast RNA metabolism.

Chloroplasts are typical organelles of photoautotrophic eukaryotic cells which drive a variety of functions, including photosynthesis. For many years the unicellular green alga Chlamydomonas reinhardtii has served as an experimental organism for studying photosynthetic processes. The recent development of molecular tools for this organism together with efficient methods of genetic analysis and the availability of many photosynthesis mutants has now made this alga a powerful model system for the analysis of chloroplast biogenesis. For example, techniques have been developed to transfer recombinant DNA into both the nuclear and the chloroplast genome. This allows both complementation tests and analyses of gene functions in vivo. Moreover, site-specific DNA recombinations in the chloroplast allow targeted gene disruption experiments which enable a "reverse genetics" to be performed. The potential of the algal system for the study of chloroplast biogenesis is illustrated in this review by the description of regulatory systems of gene expression involved in organelle biogenesis. One example concerns the regulation of trans-splicing of chloroplast mRNAs, a process which is controlled by both multiple nuclear- and chloroplast-encoded factors. The second example involves the stabilization of chloroplast mRNAs. The available data lead us predict distinct RNA elements, which interact with trans-acting factors to protect the RNA against nucleolytic attacks.

Animals↗
PubMed1985

Characterization of a Euglena gracilis chloroplast RNA polymerase specific for ribosomal RNA genes.

Euglena gracilis chloroplasts contain a 145,000-base pair chromosome that encodes genes for ribosomal, transfer, and messenger RNAs. These genes are transcribed within the organelle by chloroplast RNA polymerase activities that are specific for different classes of RNA. Two transcriptional activities have been isolated from Euglena chloroplasts. (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259, 14880-14887). One, the "soluble extract," contains enzymes active in tRNA transcription and processing. The other activity, the transcriptionally active chromosome, consisting of a chloroplast DNA-dependent RNA polymerase tightly bound to chloroplast DNA, only transcribes rRNA genes even though the entire chloroplast genome is present. We have extensively purified the transcriptionally active chromosome using high salt concentrations to dissociate loosely bound proteins. The result is a highly enriched extract containing three major polypeptides of Mr 116,000-118,000, 83,000-88,000, and 24,000-26,000 that retains complete selectivity for rDNA transcription. It is probable that one, or both, of the high molecular weight proteins are functional components of the DNA-dependent RNA polymerase. The identification and characterization of the transcriptionally active chromosome is a first step towards understanding how chloroplast rRNA synthesis is regulated.

Chloroplasts↗
PubMed1991

Highly purified pea chloroplast RNA polymerase transcribes both rRNA and mRNA genes.

Pea chloroplast RNA polymerase has been obtained with about 2000-fold purification using DEAE-cellulose and phosphocellulose chromatography. The purified enzyme contained ten prominent polypeptides of 150, 130, 115, 110, 95, 85, 75, 48, 44 and 39 kDa and four other minor polypeptides of 90, 34, 32 and 27 kDa. Purification of this enzyme using chloroplast 16S rDNA promoter affinity column chromatography also yielded an enzyme with similar polypeptides. Purified polyclonal antibodies against the purified chloroplast RNA polymerase were found to recognize most of the polypeptides of the enzyme in Western blot experiments. Primary mobility shift of the 16S rRNA gene and ribulose-1,5-bisphosphate carboxylase large subunit (rbc-L) gene promoters observed with the chloroplast RNA polymerase was abolished by these antibodies. The specific in vitro transcription of these rRNA and mRNA genes was also inhibited by these antibodies. The transcription of the rRNA and mRNA genes was also abolished by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase. The chloroplast RNA polymerase was found to bind specifically to the chloroplast 16S rRNA gene promoter region as visualized in electron microscopy. The presence of the polypeptides of 130, 110, 75-95 and 48 kDa in the DNA-enzyme complex was confirmed by a novel approach using immunogold labeling with the respective antibodies. The polypeptides of this purified RNA polymerase were found to be localized in chloroplasts by an indirect immunofluorescence.

Bacterial Toxins↗
PubMed1988

Recognition of prokaryotic transcription terminators by spinach chloroplast RNA polymerase.

To determine whether chloroplast RNA polymerase will accurately terminate transcription in vitro, we have fused the spinach chloroplast rbcL promoter to the 3' end of the rbcL gene as well as to various factor independent transcription terminators from E. coli. Transcription of the rbcL minigene did not result in production of the expected 265 nucleotide RNA. However, the spinach chloroplast RNA polymerase did terminate transcription with varying efficiency at the thra, rrnB, rrnC and gene 32 terminators. The most efficient transcription termination was observed for the threonine attenuator. For each of the prokaryotic terminators, the chloroplast enzyme ceased transcription at essentially the same position as the E. coli RNA polymerase. These data indicate that the transcription termination process in chloroplasts has some features in common with the mechanism used in prokaryotes.

Base Sequence↗
PubMed2025

A six-repeat PPR protein WPR directly binds target RNAs and coordinates chloroplast RNA processing via dual recruitment of MORF1, MORF8b, and CAF2 proteins in rice.

Pentatricopeptide repeat (PPR) proteins are key regulators of organelle RNA metabolism in plants, yet their precise mechanisms in chloroplast RNA processing remain unclear. Here, we identify WPR, a unique P-type PPR protein in rice (Oryza sativa L.), as a critical factor in chloroplast RNA splicing and editing. A ~112-kb chromosomal inversion upstream of WPR causes an albino panicle rachis phenotype (wpr mutant), while complete loss of WPR function leads to seedling lethality. WPR deficiency disrupts the splicing of multiple group II introns (atpF, ndhA, ndhB, petB, rpl2, and rps12) and impairs RNA editing in transcripts such as ndhA, ndhB, ndhG, rps14, and ycf3. Electrophoretic mobility shift assay (EMSA) data confirm that WPR directly binds to precursor mRNAs of atpF, ndhA, petB, rpl2, and rps12. Strikingly, WPR interacts with both RNA editing factors (MORF1, MORF8b) and the splicing factor CAF2, but not with other PPR proteins targeting the same transcripts. Unlike most PPR proteins, WPR contains only six PPR repeats, which is the fewest among all functionally characterized rice PPR proteins. With few informative repeats, WPR likely possesses a broad, low-specificity RNA-binding activity. Moreover, WPR may act on chloroplast RNA maturation by recruiting MORFs and CAF2 rather than other PPR proteins, highlighting a novel regulatory mode in which P-type PPR protein may act as an RNA-binding scaffold to integrate diverse RNA-processing machineries. This study advances the understanding of PPR protein diversity and provides new insights into the molecular mechanisms of chloroplast RNA processing in rice.

Oryza↗
PubMed1999

RNA editing sites in tobacco chloroplast transcripts: editing as a possible regulator of chloroplast RNA polymerase activity.

Genetic information in chloroplast DNA is sometimes altered at the transcript level by a process known as RNA editing. Sequence analysis of amplified cDNAs for 69 potential editing sites revealed 13 real editing sites in transcripts of 11 tobacco chloroplast genes. Together with those reported previously, these bring the total of edited sites observed in tobacco chloroplast transcripts to 31 (all involve C to U conversion). Alignment of sequences around the 31 editing sites revealed no obvious consensus, apart from an apparent bias for U or C at position -1 and A at position +2. Editing in tobacco rpoA mRNA restores the conserved leucine residue which is known to be important for transcriptional activation of the alpha subunit of E. coli RNA polymerase. Editing of this site is partial and the extent of editing depends on developmental conditions, suggesting that editing is, at least in part, involved in the regulation of chloroplast-encoded RNA polymerase activity.

Amino Acid Sequence↗
PubMed1994

Evidence that sigma factors are components of chloroplast RNA polymerase.

Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae.

Animals↗
PubMed2006

A pentatricopeptide repeat protein is a site recognition factor in chloroplast RNA editing.

In higher plants, RNA editing is a post-transcriptional process that converts C to U in organelle mRNAs. We have previously shown that an Arabidopsis thaliana crr4 mutant is defective with respect to RNA editing for creating the translational initial codon of the plastid ndhD gene (the ndhD-1 site). CRR4 contains 11 pentatricopeptide repeat motifs but does not contain any domains that are likely to be involved in the editing activity. The green fluorescent protein fused to the putative transit peptide of CRR4 targeted the plastid. The recombinant CRR4 expressed in Escherichia coli specifically bound to the 25 nucleotides of the upstream and the 10 nucleotides of the downstream sequences surrounding the editing site of ndhD-1. The target C nucleotide of this editing is not essential for the binding of CRR4. Taken together with the genetic evidence, we conclude that the pentatricopeptide repeat protein CRR4 is a sequence-specific RNA-binding protein that acts as a site recognition factor in plastid RNA editing.

Arabidopsis↗
PubMed2004

Roles of chloroplast RNA polymerase sigma factors in chloroplast development and stress response in higher plants.

Chloroplast transcription in higher plants is performed by two types of RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is a eubacteria-type multisubunit enzyme whose catalytic core subunits are encoded by the chloroplast genome, whereas NEP is the nuclear encoded T7 phage-type single subunit enzyme. PEP is critical for the biogenesis and maintenance of chloroplasts, and is finely tuned by the nuclear encoded sigma subunits. Of the six Arabidopsis sigma subunits, SIG2 is involved in the transcription of several chloroplast tRNA genes, including trnE encoding tRNA-Glu. SIG2 possibly couples translation and pigment synthesis in chloroplasts. On the other hand, SIG5 is induced by various stresses and contributes to repair of damaged photosystem II (PSII) through transcription of the psbD and psbC genes. Thus target genes and the physiological role of each sigma subunit are becoming clearer.

Base Sequence↗
PubMed1996

Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.

We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level.

Amino Acid Sequence↗
PubMed1995

Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences.

We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli. Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied. We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency. The sequence of the G+C stem-loop itself also influences termination. Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not. From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner. The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.

Base Composition↗
PubMed1997

Transcription factor phosphorylation by a protein kinase associated with chloroplast RNA polymerase from mustard (Sinapis alba).

The chloroplast transcription machinery involves multiple components with both catalytic and regulatory functions. Here we describe a serine-specific protein kinase activity that is associated with the major chloroplast RNA polymerase and phosphorylates sigma-like transcription factors in vitro. The kinase activity can be assigned to a 54 kDa polypeptide of partially purified RNA polymerase (KPC, kinase polymerase complex). This polypeptide is also present in a smaller complex that contains several putative polymerase subunits and reveals kinase activity but lacks transcription activity (KC, kinase complex). Although the 54 kDa component could not be chromatographically separated from the rest of this complex without loss of activity, it retained residual kinase activity in an electrophoretic blot assay. The polymerase-associated kinase is itself affected by in vitro phosphorylation and dephosphorylation, which raises the possibility that it is part of a signalling cascade that controls chloroplast transcription in vivo by factor phosphorylation.

Chloroplasts↗
PubMed1980

Properties and characterization of a spinach chloroplast RNA polymerase isolated from a tanscriptionally active DNA-protein complex.

A chloroplast RNA polymerase has been isolated from a transcriptionally active spinach plastid DNA-protein complex. The properties of the complex and of the reconstituted system have been compared. The crude enzyme is at least sevenfold less active when compared with the complex. RNA synthesis by the reconstituted system is sensitive to high ionic strength and heparin, contrarily to RNA synthesis by the chloroplast DNA-protein complex. On the other hand, rifampicin has no inhibitory effect whatever on the transcriptional system used. The RNA polymerase isolated is more efficient with denatured DNA than with double-stranded DNA and the best template is chloroplast DNA. The crude RNA polymerase isolated migrates in a peak of 11 S in glycerol gradient centrifugation and is located in a single band in non-denaturing polyacrylamide gel electrophoresis. About 30 polypeptides (Mr 15 000--180 000) are part of the complex and only eight of them are found in the RNA polymerase preparation. Only five polypeptides are always present with the same yield. They are probably the subunits of the RNA polymerase. The molecular weight of these subunits ranged from 15 000--69 000, even if the isolation of the enzyme was performed in the presence of protease inhibitors.

Chloroplasts↗
PubMed1980

Preferential transcription of cloned maize chloroplast DNA sequences by maize chloroplast RNA polymerase.

Zea mays chloroplast DNA-dependent RNA polymerase in vitro preferentially transcribes maize chloroplast DNA sequences incorporated in cloned chimeric bacterial plasmids. Preferential transcription is dependent on the presence of a 27.5-kilodalton polypeptide, the S factor, which has been purified from maize chloroplasts, and also on the template's being in the supercoiled form.

Journal Article↗
PubMed1996

Nuclear encoding of a chloroplast RNA polymerase sigma subunit in a red alga.

A chloroplast RNA polymerase sigma factor is encoded by a nuclear gene, sigA, in the red alga Cyanidium caldarium RK-1. The encoded protein functions as an RNA polymerase sigma factor in vitro and it is localized to the chloroplast in vivo. SigA shows high sequence similarity to the sigma factors of cyanobacteria, which is indicative of the ancestral endosymbiotic event and subsequent transfer of the sigA gene to the nuclear genome.

Amino Acid Sequence↗
PubMed1980

A facile procedure for purifying maize chloroplast RNA polymerase from whole cell homogenates.

Two DNA-dependent RNA polymerases have been purified from homogenates of maize leaves by a relatively rapid procedure involving Sepharose 4B and DEAE-cellulose chromatography followed by resolution of two RNA polymerasies on phosphocellulose. The RNA polymerase eluting from phosphocellulose at 0.11 M (NH4)2SO4 is inhibited strongly by low levels of alpha-amanitin and possesses catalytic properties and polypeptide subunits like those of maize nuclear RNA polymerase II. The RNA polymerase eluting from phosphocellulose at 0.15 M (NH4)2SO4 resembles the RNA polymerase solubilized from isolated maize chloroplasts in many ways. This enzyme and that isolated from chloroplasts are resistant to alpha-amanitin and rifamycin-SV at high concentrations. Both RNA polymerases have virtually the same Mn2+ and Mg2+ optima, Mg2+/Mn2+ activity ratios, (NH4)2SO4 sensitivity, kinetics of UMP incorporation, and temperature optima. Electrophoresis of this phosphocellulose-purified RNA polymerase on denaturing polyacrylamide slab gels reveals 14 heavily stainable polypeptides that are identical in number and molecular mass to those from chloroplast RNA polymerase. Moreover, two-dimensional tryptic maps of the 14 polypeptides from the phosphocellulose-purified RNA polymerase are very similar to the maps of corresponding polypeptides from chloroplast RNA polymerase. Using this method, relatively large quantities (0.5 mg/kg leaves) of a form of chloroplast RNA polymerase can be prepared in a few days.

Centrifugation, Density Gradient↗