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Query: EC:3.1.27.1 (RNase)
 16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chlamydomonas reinhardtii cells treated with toluene at 0 degrees C and 25 degrees C incorporate ribonucleoside triphosphates (NTPs) into chloroplast RNA at 25 degrees C and also at 35 degrees C. The incorporation requires all four NTPs and Mg2+, and is completely inhibited by DNase, RNase, actinomycin D (40 microgram/ml) and rifampicin (350 microgram/ml). However, the incorporation is almost totally insensitive to both alpha-amanitin and streptolydigin at 200 microgram/ml.
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PMID:Synthesis of chloroplast ribonucleic acid in Chlamydomonas reinhardtii toluene-treated cells. 25 50

Over half of the chloroplast ribosomes isolated from growing cultures of Chlamydomonas reinhardtii are bound to chloroplast thylakoid membranes if completion of nascent polypeptide chains is prevented by chloramphenicol. The free chloroplast ribosomes are recovered in homogenate supernatants, and presumably originate from the chloroplast stroma. Only about 10% of these free chloroplast ribosomes are polyribosomes, even under conditions when 70% of free cytoplasm ribosomes are recovered as polyribosomes. The nonionic detergent Nonidet P-40 liberates atypical polyribosomes (Type I), from membranes, which require both ribonuclease and proteases for complete conversion to monomeric ribosomes. Thus Type I particles are held together by mRNA but are also held together by peptide bonds. These Type I polyribosomes probably are not bound to intact membrane, but might be bound to some protein-containing sub-membrane particle. The Type I polyribosomes are dissociated to ribosomal subunits by puromycin and high salt, and contained 0.2 to 1 nascent chain per ribosome. If membranes are treated with Nonidet and proteases at the same time, polyribosomes which are digested to monomeric ribosomes by ribonuclease alone (Type II) are obtained. Type II polyribosomes are smaller than Type I, and probably represent the true size distribution of polyribosomes on the membranes. At least 50% of the membrane-bound ribosomes are polyribosomes, since that much membrane bound chloroplast RNA is recovered as Type I or Type II polyribosomes.
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PMID:Free and membrane-bound chloroplast polyribosomes Chlamydomonas reinhardtii. 116 19

The formation of a stable complex between glutamyl-tRNA synthetase and the first enzyme of chlorophyll biosynthesis glutamyl-tRNA reductase was investigated in the green alga Chlamydomonas reinhardtii. Apparently homogenous enzymes, purified after previously established purification protocols were incubated in various combinations with ATP, glutamate, tRNA(Glu) and NADPH and formed complexes were isolated via glycerol gradient centrifugation. Stable complexes were detected only after the preincubation of glutamyl-tRNA synthetase, glutamyl-tRNA reductase with either glutamyl-tRNA or free tRNA(Glu), ATP and glutamate, indicating the obligatory requirement of aminoacylated tRNA(Glu) for complex formation. The further addition of NADPH resulting in the reduction of the tRNA-bound glutamate to glutamate 1-semialdehyde led to the dissociation of the complex. Once complexed to the two enzymes tRNA(Glu) was found to be partially protected from ribonuclease digestion. Escherichia coli, Bacillus subtilis and Synechocystis 6803 tRNA(Glu) were efficiently incorporated into the protein-RNA complex. The detected complexes provide the chloroplast with a potential channeling mechanism for Glu-tRNA(Glu) into chlorophyll synthesis in order to compete with the chloroplastic protein synthesis machinery.
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PMID:Complex formation between glutamyl-tRNA synthetase and glutamyl-tRNA reductase during the tRNA-dependent synthesis of 5-aminolevulinic acid in Chlamydomonas reinhardtii. 145 6

Using a modified vector, we developed a method for DNA-mediated transformation of Chlamydomonas reinhardi with increased efficiency. The vector contained the yeast 2 microns origin of replication as a heterologous replicon. The aminoglycoside 3'-phosphotransferase (APH) gene linked to the simian virus 40 early promoter was used as an antibiotic selectable marker. The C. reinhardi transformants were resistant to 12 micrograms of G418 or 150 micrograms of kanamycin per ml. A quick-blot mRNA analysis demonstrated the presence of RNase-sensitive transcripts from the APH gene in the transformants, suggesting that the acquisition of antibiotic resistance was due to the expression of the APH gene. Southern blot analysis revealed the presence of free plasmid DNA in the transformant. The transforming vector was recovered by transforming recipient bacteria with the total DNA extracted from the C. reinhardi transformant.
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PMID:DNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3'-phosphotransferase as a selectable marker. 301 25

The amount of chloroplast ribosomal RNAs of Chlamydomonas reinhardtii which sediment at 15,000 g is increased when cells are treated with chloramphenicol. Preparations of chloroplast membranes from chloramphenicol-treated cells contain more chloroplast ribosomal RNAs than preparations from untreated cells. The membranes from treated cells also contain more ribosome-like particles, some of which appear in polysome-like arrangements. About 50% of chloroplast ribosomes are released from membranes in vitro as subunits by 1 mM puromycin in 500 mM KCl. A portion of chloroplast ribosomal subunits is released by 500 mM KCl alone, a portion by 1 mM puromycin alone, and a portion by 1 mM puromycin in 500 mM KCl. Ribosomes are not released from isolated membranes by treatment with ribonuclease. Membranes in chloroplasts of chloramphenicol-treated cells show many ribosomes associated with membranes, some of which are present in polysome-like arrangements. This type of organization is less frequent in chloroplasts of untreated cells. Streptogramin, an inhibitor of initiation, prevents chloramphenicol from acting to permit isolation of membrane-bound ribosomes. Membrane-bound chloroplast ribosomes are probably a normal component of actively growing cells. The ability to isolate membrane-bound ribosomes from chloramphenicol-treated cells is probably due to chloramphenicol-prevented completion of nascent chains during harvesting of cells. Since chloroplasts synthesize some of their membrane proteins, and a portion of chloroplast ribosomes is bound to chloroplast membranes through nascent protein chains, it is suggested that the membrane-bound ribosomes are synthesizing membrane protein.
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PMID:Ribosomes bound to chloroplast membranes in Chlamydomonas reinhardtii. 480 47

Toward understanding regulation of chloroplast transcript abundance, we have isolated and analyzed nuclear mutant strains of Chlamydomonas reinhardtii that lack chloroplast-encoded mRNAs for photosystem II proteins. Mutant 6.2z5 accumulates no transcripts of the psbC locus for the 43-kilodalton chlorophyll-binding protein. In mutant GE2.10, transcripts of psbB, encoding the 47-kilodalton chlorophyll-binding protein, cannot be detected [Jensen, K.H., Herrin, D.L., Plumley, F.G., and Schmidt, G.W. (1986). J. Cell Biol. 103, 1315-1325]. Also, GE2.10 does not accumulate several low molecular weight transcripts from a region of the chloroplast genome proximal to psbB. The levels of mRNAs from other chloroplast genes are not affected in either mutant. Chloroplast transcription was analyzed in permeabilized cells and by in vivo pulse labeling. Although 5[prime] ribonuclease was found as an artifactual activity of permeabilized cells, the results from both assays demonstrated that wild-type levels of psbC transcription occur in mutant 6.2z5 and that chloroplasts of GE2.10 transcribe psbB and adjacent genes. Thus, it appears that the nuclear genes that are mutated in 6.2z5 and GE2.10 encode products that, respectively, confer stability to transcripts from the psbC and the psbB regions of the chloroplast genome.
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PMID:Chloroplast RNA Stability in Chlamydomonas: Rapid Degradation of psbB and psbC Transcripts in Two Nuclear Mutants. 1232 94

Cell survival depends on the cell's ability to acclimate to phosphorus (P) limitation. We studied the chloroplast ribonuclease polynucleotide phosphorylase (PNPase), which consumes and generates phosphate, by comparing wild-type Chlamydomonas reinhardtii cells with strains with reduced PNPase expression. In the wild type, chloroplast RNA (cpRNA) accumulates under P limitation, correlating with reduced PNPase expression. PNPase-deficient strains do not exhibit cpRNA variation under these conditions, suggesting that in the wild type PNPase limits cpRNA accumulation under P stress. PNPase levels appear to be mediated by the P response regulator PHOSPHORUS STARVATION RESPONSE1 (PSR1), because in psr1 mutant cells, cpRNA declines under P limitation and PNPase expression is not reduced. PNPase-deficient cells begin to lose viability after 24 h of P depletion, suggesting that PNPase is important for cellular acclimation. PNPase-deficient strains do not have enhanced sensitivity to other physiological or nutrient stresses, and their RNA and cell growth phenotypes are not observed under P stress with phosphite, a phosphate analog that blocks the stress signal. In contrast with RNA metabolism, chloroplast DNA (cpDNA) levels declined under P deprivation, suggesting that P mobilization occurs from DNA rather than RNA. This unusual phenomenon, which is phosphite- and PSR1-insensitive, may have evolved as a result of the polyploid nature of cpDNA and the requirement of P for cpRNA degradation by PNPase.
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PMID:Integration of chloroplast nucleic acid metabolism into the phosphate deprivation response in Chlamydomonas reinhardtii. 1735 Nov 18

We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.
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PMID:MRL1, a conserved Pentatricopeptide repeat protein, is required for stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis. 2009 72

mRNA levels result from an equilibrium between transcription and degradation. Ribonucleases (RNases) facilitate the turnover of mRNA, which is an important way of controlling gene expression, allowing the cells to adjust transcript levels to a changing environment. In contrast to the heterotrophic model bacteria Escherichia coli and Bacillus subtilis, RNA decay has not been studied in detail in cyanobacteria. Synechocystis sp. PCC6803 encodes orthologs of both E. coli and B. subtilis RNases, including RNase E and RNase J, respectively. We show that in vitro Sy RNases E and J have an endonucleolytic cleavage specificity that is very similar between them and also compared to orthologous enzymes from E. coli, B. subtilis, and Chlamydomonas. Moreover, Sy RNase J displays a robust 5'-exoribonuclease activity similar to B. subtilis RNase J1, but unlike the evolutionarily related RNase J in chloroplasts. Both nucleases are essential and gene deletions could not be fully segregated in Synechocystis. We generated partially disrupted strains of Sy RNase E and J that were stable enough to allow for their growth and characterization. A transcriptome analysis of these strains partially depleted for RNases E and J, respectively, allowed to observe effects on specific transcripts. RNase E altered the expression of a larger number of chromosomal genes and antisense RNAs compared to RNase J, which rather affects endogenous plasmid encoded transcripts. Our results provide the first description of the main transcriptomic changes induced by the partial depletion of two essential ribonucleases in cyanobacteria.
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PMID:Impact of RNase E and RNase J on Global mRNA Metabolism in the Cyanobacterium Synechocystis PCC6803. 3258 60