Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently determined the nucleotide sequence of the gene encoding p28, a light chain of inner dynein arms of Chlamydomonas axonemes. Here, we show that p28 is the protein encoded by the IDA4 locus. p28, and the dynein heavy chains normally associated with it, are completely absent from the flagella and cell bodies of three allelic strains of ida4, named ida4-1, ida4-2, and ida4-3. We determined the nucleotide sequence of the three alleles of the p28 gene and found in each case a single nucleotide change, affecting the splice sites of the first, second, and fourth introns, respectively. Reverse transcriptase-polymerase chain reaction amplification of RNAs prepared from ida4 cells confirmed that these mutations prevent the correct splicing of the affected introns, thereby blocking the synthesis of full-length p28. These are the first intron splicing mutations described in Chlamydomonas and the first inner dynein arm mutations characterized at the molecular level. The absence in ida4 axonemes of the dynein heavy chains normally found in association with p28 suggests that p28 is necessary for stable assembly of a subset of inner dynein arms or for the binding of these arms to the microtubule doublets.
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PMID:ida4-1, ida4-2, and ida4-3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms. 757 90

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.
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PMID:Evidence that sigma factors are components of chloroplast RNA polymerase. 815 91

The chloroplast genome consists of homogeneous circular DNA molecules. To date, the entire nucleotide sequences (120-190 kbp) of chloroplast genomes have been determined from eight plant species. The chloroplast genomes of land plants and green algae contain about 110 different genes, which can be classified into two main groups: genes involved in gene expression and those related to photosynthesis. The red alga Porphyra chloroplast genome has 70 additional genes, one-third of which are related to biosynthesis of amino acids and other low molecular mass compounds. Chloroplast genes contain at least three structurally distinct promoters and transcribe two or more classes of RNA polymerase. Two chloroplast genes, rps12 of land plants and psaA of Chlamydomonas, are divided into two to three pieces and scattered over the genome. Each portion is transcribed separately, and two to three separate transcripts are joined together to yield a functional mRNA by trans-splicing. RNA editing (C to U base changes) occurs in some of the chloroplast transcripts. Most edited codons are functionally significant, creating start and stop codons and changing codons to retain conserved amino acids.
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PMID:The chloroplast genome. 882 48

The biosynthesis of the hemes, chlorophylls, corrins and other tetrapyrroles begins with the synthesis of 5-aminolevulinic acid (ALA). The pathway is highly conserved except for the synthesis of ALA which is derived from glycine and succinyl CoA (C4) in most eukaryotes and from glutamate (C5) in most bacteria and in green plants. In C5, glutamyl-tRNA synthetase (GTS) converts glutamate to glutamyl-tRNA (glu-tRNA), which is reduced by glutamyl-tRNA reductase (GTR) to glutamyl-1-semialdehyde (GSA), which is converted by aminotransferase (GSA-AT) to ALA. Since GTS is also involved in protein synthesis and GSA can be converted to ALA non-enzymatically, it is highly probable that control of ALA synthesis and thus of the whole pathway resides in the GTR step. In Escherichia coli, GTR is the gene product of hemA. BL21(DE3), a protease-deficient strain which contains the T7 RNA polymerase gene in front of a lac promoter, was transformed with a pET14b-based vector, pWC01, harboring hemA in front of a T7 promoter and ORF1 which is transcribed in the opposite direction. The transformed strain, WC1201, secreted ALA and porphyrins into the medium. Induction of expression of hemA by WC1201 was optimized for concentration of inducer (IPTG, 5 mM), temperature (37 degrees C), presence of betaine and sorbitol (no change) and time of induction (2h). GTR was observable as a 46 kDa band by Brilliant blue G staining of SDS-PAGE gels. Sonicates of the induction mixture exhibited strong ALA synthesis activity which was enhanced by tRNAglu. Most of the activity was in the supernatant of the sonicate indicating that GTR is a soluble enzyme. The induced strain had more GTS activity than the uninduced strain which had more GTS activity than its parent wild-type strain. Autoradiography on native gradient PAGE showed that GTR expressed in vivo by induction of WC1201 had a molecular weight of approx. 117 kDa. Gel filtration of the induced sonicate showed a peak of enzymatic activity at about 126 kDa. When pET14b- or pUC19-based plasmids harboring hemA and ORF1, or importantly, a pUC19-based plasmid harboring only hemA and not ORF1, were expressed in an in vitro transcription-translation system, native gradient PAGE showed a product with a molecular weight of approximately 175 kDA. This expression was higher in the presence of tRNAglu. When the 117 kDa and 175 kDa proteins were excised from their native gels respectively, and run on SDS PAGE, autoradiography showed bands at 46 kDa. We conclude that GTR is present in both high molecular weight species. Since overexpression of hemA from pET14b-based plasmids is associated with increased glutamyl-tRNA synthetase activity, the 175 kDa species may represent different complexes of GTR, GTS and glutamyl-tRNA as observed in Chlamydomonas and the 117-126 kDa species may be an dimer of GTR associated with glu-tRNA or a complex of GTR, GTS and glu-tRNA. These possibilities are being investigated.
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PMID:Expression of glutamyl-tRNA reductase in Escherichia coli. 895 Jan 86

A U3 snoRNA gene isolated from a Chlamydomonas reinhardtii (CRE:) genomic library contains putative pol III-specific transcription signals similar to those of RNA polymerase III-specific small nuclear (sn)RNA genes of higher plants. The 222 nt long CRE: U3 snoRNA was immunoprecipitated by anti-gamma-mpppN antisera, but not by anti-m(2,2,7)G antibodies, supporting the notion that it is a RNA polymerase III transcript. Tagged CRE: U3 snoRNA gene constructs were expressed in CRE: cells. Results of chemical and enzymatic structure probing of CRE: U3 snoRNA in solution and of DMS modification of CRE: U3 snoRNA under in vivo conditions revealed that the two-hairpin structure of the 5'-domain that is found in solution is no longer detected under in vivo conditions. The observed differences can be explained by the formation of several base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. A model that involves five intermolecular helices is proposed.
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PMID:Molecular characterization at the RNA and gene levels of U3 snoRNA from a unicellular green alga, Chlamydomonas reinhardtii. 1090 60

Chlamydomonas reinhardtii is a unicellular eukaryotic alga possessing a single chloroplast that is widely used as a model system for the study of photosynthetic processes. This report analyzes the surprising structural and evolutionary features of the completely sequenced 203,395-bp plastid chromosome. The genome is divided by 21.2-kb inverted repeats into two single-copy regions of approximately 80 kb and contains only 99 genes, including a full complement of tRNAs and atypical genes encoding the RNA polymerase. A remarkable feature is that >20% of the genome is repetitive DNA: the majority of intergenic regions consist of numerous classes of short dispersed repeats (SDRs), which may have structural or evolutionary significance. Among other sequenced chlorophyte plastid genomes, only that of the green alga Chlorella vulgaris appears to share this feature. The program MultiPipMaker was used to compare the genic complement of Chlamydomonas with those of other chloroplast genomes and to scan the genomes for sequence similarities and repetitive DNAs. Among the results was evidence that the SDRs were not derived from extant coding sequences, although some SDRs may have arisen from other genomic fragments. Phylogenetic reconstruction of changes in plastid genome content revealed that an accelerated rate of gene loss also characterized the Chlamydomonas/Chlorella lineage, a phenomenon that might be independent of the proliferation of SDRs. Together, our results reveal a dynamic and unusual plastid genome whose existence in a model organism will allow its features to be tested functionally.
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PMID:The Chlamydomonas reinhardtii plastid chromosome: islands of genes in a sea of repeats. 1241 94

The Chlamydomonas reinhardtii plastid and mitochondrial transcriptomes were surveyed for changes in RNA profiles resulting from growth in 12 culture conditions representing 8 abiotic stimuli. Organellar RNA abundance exhibited marked changes during nutrient stress and exposure to UV light, as revealed by both RNA gel blot and DNA microarray analyses. Of particular note were large increases in tufA and clpP transcript abundance during nutrient limitation. Phosphate and sulfur limitation resulted in the most global, yet opposite, effects on organellar RNA abundance, changes that were dissected further using run-on transcription assays. Removal of sulfate from the culture medium, which is known to reduce photosynthesis, resulted in 2-fold to 10-fold decreases in transcription rates, which were reflected in lower RNA abundance. The decrease in transcriptional activity was completely reversible and recovered to twice the control level after sulfate replenishment. Conversely, phosphate limitation resulted in a twofold to threefold increase in RNA abundance that was found to be a post-transcriptional effect, because it could be accounted for by increased RNA stability. This finding is consistent with the known metabolic slowdown under phosphate stress. Additionally, inhibitor studies suggested that unlike those in higher plants, Chlamydomonas chloroplasts lack a nucleus-encoded plastid RNA polymerase. The apparently single type of polymerase could contribute to the rapid and genome-wide transcriptional responses observed within the chloroplast.
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PMID:The Chlamydomonas reinhardtii organellar genomes respond transcriptionally and post-transcriptionally to abiotic stimuli. 1595 33

In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors, and a nuclear-encoded phage-like RNAP. Green algae, however, appear to possess only the bacterial enzyme. Since transcription of much, if not most, of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus, we sought to identify sigma factor genes that might be responsible for this regulation. We describe a nuclear gene (RPOD) that is predicted to encode an 80 kDa protein that, in addition to a predicted chloroplast transit peptide at the N-terminus, has the conserved motifs (2.1- 4.2) diagnostic of bacterial sigma-70 factors. We also identified two motifs not previously recognized for sigma factors, adjacent PEST sequences and a leucine zipper, both suggested to be involved in protein-protein interactions. PEST sequences were also found in approximately 40% of sigma factors examined, indicating they may be of general significance. Southern blot hybridization and BLAST searches of the genome and EST databases suggest that RPODmay be the only sigma factor gene in C. reinhardtii. The levels of RPODmRNA increased 2- 3-fold in the mid-to-late dark period of light-dark cycling cells, just prior to, or coincident with, the peak in chloroplast transcription. Also, the dark-period peak in RPOD mRNA persisted in cells shifted to continuous light or continuous dark for at least one cycle, indicating that RPODis under circadian clock control. These results suggest that regulation of RPODexpression contributes to the circadian clock's control of chloroplast transcription.
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PMID:Structure, circadian regulation and bioinformatic analysis of the unique sigma factor gene in Chlamydomonas reinhardtii. 1614 45

Chlamydomonas reinhardtii EST clones encoding a protein highly similar to prokaryotic sigma factors and plant sigma-like factors (SLFs) were used to isolate a BAC clone containing the full-length gene CrRpoD. The gene is likely to be single-copy, in contrast to small gene families encoding SLFs in plants. The CrRpoD mRNA comprises 3,033 nt with an open reading frame of 2,256 nt, encoding a putative protein of 752 amino acids with a molecular mass of 80.2 kDa. The sequence contains conserved regions 2-4 typically found in sigma factors, and an unusually long amino terminal extension, which by in silico analysis has properties of a chloroplast transit peptide. Expression of CrRpoD was confirmed by immunodetection of a 85 kDa polypeptide in a preparation enriched for chloroplast proteins. To demonstrate functionality in transcription initiation, a recombinant CrRpoD-thioredoxin fusion protein was reconstituted with E. coli RNA polymerase core enzyme and tested in vitro. This chimeric holoenzyme specifically bound the spinach psbA and Chlamydomonas rrn16 promoters in gel mobility shift assays and exhibited specific transcription initiation from the same two promoters, providing evidence for the role of CrRpoD as a functional transcription factor.
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PMID:Chlamydomonas reinhardtii encodes a single sigma70-like factor which likely functions in chloroplast transcription. 1645 12

The inhibitory activities of amatoxins on the growth of Chlamydomonas reinhardtii have been determined using a convenient assay based upon incubation in multiwell tissue culture plates followed by turbidimetric estimates of growth on a multiwell plate reader. Values for the inhibitory dosage at which growth is 50% of untreated culture (ID(50)) of 5.4, 6.6, and 5.6 micromolar were obtained for alpha-amanitin, O-methyl-alpha-amanitin, and amaninamide, respectively. Treatment of liquid cultures with 1 microgram per milliliter N-methyl-N' -nitro-N-nitrosoguanidine followed by growth in agar pour tubes containing 25 micromolar alpha-amanitin led to the selection of several lines demonstrating varying resistance to amanitin inhibition, with ID(50) values from 36 micromolar to greater than 200 micromolar. Two lines completely resistant to inhibition by 200 micromolar alpha-amanitin provided partially purified RNA polymerase activities that were 160-fold and 5600-fold more resistant to inhibition than the analogous enzyme activity from the wild-type strain. These studies provide evidence that Chlamydomonas reinhardtii does not contain significant activity capable of inactivating alpha-amanitin and that this amatoxin may be used to select for RNA polymerase mutants.
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PMID:Isolation of Amatoxin-Resistant Lines of Chlamydomonas reinhardtii: Evidence for RNA Polymerase Mutants. 1666 20


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