Synthesis of beta-galactosidase messenger RNA during amino acid starvation of stringent and relaxed strains of Escherichia coli.
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Biomedical subjects
Publications and source records attributed to M Artman.
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A method is described for selective isolation of mutants deficient in CAP protein necessary for the expression of catabolite-sensitive operons in Escherichia coli.
Cyclic guanosine 3',5'-monophosphate inhibits the synthesis of beta-galactosidase and tryptophanase in cultures of Escherichia coli growing in minimal media with glucose or glycerol as the carbon source. Cyclic guanosine 3',5'-monophosphate acts at the transcriptional level in the lac operon, it exerts its action at the promoter site of the operon, and requires the presence of functional cyclic adenosine 3',5'-monophosphate receptor protein.
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Studies were made of the synthesis of beta-galactosidase by Escherichia coli in the presence of 5-fluorouracil, streptomycin, and subinhibitory concentrations of chloramphenicol. The preferential inhibition of beta-galactosidase synthesis observed in the presence of the above drugs was found to be caused by catabolite repression.
Studies were made of the synthesis of Lac messenger ribonucleic acid (mRNA) by Escherichia coli in the absence of protein synthesis and of the coupling of transcription of lac operon to translation. Lac mRNA was not synthesized in the presence of chloramphenicol, and its synthesis steadily decreased during K(+) deprivation and treatment with puromycin. Since under these conditions total mRNA synthesis is not inhibited it is suggested that the control of Lac mRNA is distinct from that which regulates total mRNA synthesis. Lac mRNA synthesized during recovery from K(+) starvation or from chloramphenicol inhibition is not translated into functional enzyme, suggesting translational control over beta-galactosidase synthesis.
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A simple and efficient method for hybridization and subsequent recovery of non-fragmented ribosomal RNA from the hybrid is described. The procedure involves annealing of immobilized denatured DNA bound on cellulose nitrate membrane filters to complementary RNA in 50% (v/v) formamide-0.33m-potassium chloride-10mm-tris-hydrochloric acid buffer, pH7.4, at 33 degrees for 3hr. Under these conditions no detectable changes in the sedimentation coefficients of the input RNA were detected. The RNA can subsequently be recovered quantitatively from the hybrid in intact form by incubating the filters in formamide or in 85% (v/v) dimethyl sulphoxide. The applicability of the method for the evaluation of the absolute size of ribosomal RNA cistrons in Escherichia coli DNA and for the determination of the size of messenger RNA molecules is discussed.
1. RNA synthesized by Escherichia coli during one-hundredth of the generation time contains two fractions distinguishable by hybridization with homologous DNA. One fraction, approximately 30% of the newly synthesized RNA, did not compete with ribosomal RNA, being apparently messenger RNA. The other fraction, approximately 70% of the newly made RNA, hybridized as ribosomal RNA. These values are comparable with previous estimates (McCarthy & Bolton, 1964; Pigott & Midgley, 1968). 2. Hybridization-competition experiments showed that the newly made RNA associated with 70s ribosomes and larger ribosome aggregates was a mixture of ribosomal RNA and messenger RNA, whereas that associated with nascent ribosomal subunits consisted exclusively of ribosomal RNA. This observation provides means by which newly synthesized ribosomal RNA can be isolated free from messenger RNA. 3. Newly made ribosomal RNA in nascent ribosomal subunits was sensitive to shear under conditions where ribosomal RNA in mature ribosomes was shear-resistant. Thus, when RNA was extracted from cells of E. coli disrupted by mechanical means, newly made ribosomal RNA appeared heterogeneous in size, sedimenting as a broad peak extending from 8s to 16s. 4. Newly synthesized ribosomal RNA in nascent ribosomal subunits was rapidly degraded in the presence of actinomycin D and during glucose starvation. 5. Newly synthesized ribosomal RNA stimulated amino acid incorporation in a system synthesizing protein in vitro to the same extent as the RNA which contained the messenger RNA fraction.
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1. Ribonuclease II of Escherichia coli degrades pulse-labelled RNA associated with ribosomes and polyuridylic acid on ribosomes and in solution to mononucleotides. 2. Ribosomal and pulse-labelled RNA in solution and ribosomal RNA in chloramphenicol particles (protein-deficient ribosomes) are degraded to oligonucleotides. 3. Ribosomal RNA in mature ribosomes is not attacked by the enzyme. 4. From the mode of action of ribonuclease II, which is specific for single-stranded polyribonucleotides and does not attack helical forms, it is inferred that pulse-labelled RNA associated with ribosomes of E. coli exists as a single-stranded structure and that ribosomal RNA in chloramphenicol particles has a pronounced helical character. 5. The different behaviour of ribonuclease II towards newly synthesized RNA, ribosomal RNA and chloramphenicol-particle RNA in E. coli ribosomes is discussed.
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