Polyribosomes in Azotobacter vinelandii

Oppenheim, Joel D.; Scheinbuks, Julian; Biava, Claude G.; Marcus, Leon

doi:10.1016/0005-2787(68)90117-2

Cited by 27 publications

(4 citation statements)

References 20 publications

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“…This in vitro demonstration confirms a conclusion already indicated by the finding that bacterial cells incubated at 0°convert their polysomes into ribosomes (Goldstein et al, 1964;Das and Goldstein, 1968;Oppenheim et al, 1968). (These experiments were the basis for the use of slow cooling for accumulating runoff ribosomes in cells: Algranati et al, 1969;Subramanian et al, 1969.…”

Section: Discussionsupporting

confidence: 89%

Properties of initiation-free polysomes of Escherichia coli

Tai¹,

Wallace²,

Herzog³

et al. 1973

Biochemistry

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Purified polysomes were obtained from gently prepared lysates of Escherichia coli and from ribosomes incubated with phage R17 RNA, by gel filtration on Sepharose 4B. The endogenous polysomes incorporated amino acids several times as fast as the R17 viral polysomes. Most preparations lacked initiating activity: incorporation was complete WJtudies in peptide synthesis with natural messenger have made use either of bacterial extracts with added viral messenger (see review by Kozak and Nathans, 1972) or of gently prepared cell lysates containing endogenous polysomes (Staehelin et al., 1963;Godson and Sinsheimer, 1967;Pestka and Hintikka, 1971). However, these systems support chain initiation as well as chain elongation, and to analyze mechanisms of interference with protein synthesis (e.g., by antibiotics) it would be desirable to compare such preparations with a polysome preparation that carried out only chain elongation. Moreover, polysomes made with viral as well as with cellular messenger would be useful for comparing an initiating and a noninitiating system employing the same messenger (viral RNA), and also for verifying the relevance, for the cell, of antibiotic mechanisms observed with this messenger in vitro.This paper will describe the preparation of active purified polysomes of Escherichia coli that lack initiation factors (IF),* 1 and will compare the response of these preparations to changes in Mg2+ concentration and temperature with the response of an initiating system. The use of the initiating and noninitiating systems to study the actions of aurintricarboxylate, kasugamycin, and pactamycin will be described in the following paper (Tai et al., 1973). Similar studies have revealed unexpected specific effects of streptomycin, spectinomycin, and erythromycin on an initiating system, which will be reported in subsequent publications. Materials and MethodsBacterial Strains and Growth. MRE600, an RNase I-E. coli strain (Cammack and Wade, 1965), was used for the preparation of endogenous polysomes, supernatant fractions, and crude IF. For preparing viral polysomes in vitro, E. coli K12 strain s26 (Garen and Siddiqui, 1962) was used since S30 extracts (Nirenberg and Matthaei, 1961) of this strain formed more polysomes than did extracts of MRE600. Cells were grown at 37°with vigorous aeration in medium A (Davis and Mingioli, 1950) supplemented with CaCl2 (5 mg/1.), FeS04 (250 /xg/1.), glucose (0.4%), and Difco Casamino acids

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Section: Discussionsupporting

confidence: 89%

Properties of initiation-free polysomes of Escherichia coli

Tai¹,

Wallace²,

Herzog³

et al. 1973

Biochemistry

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“…Recently, Oppenheim et al 8 have obtained similar results with Azotobacter vinelandii disrupted very gently by direct osmotic shock.…”

supporting

confidence: 65%

PHYSIOLOGICAL ROLE OF 70 S RIBOSOMES IN BACTERIA

Algranati

González

Bade

1969

Proc. Natl. Acad. Sci. U.S.A.

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Abstract.-Evidence is presented indicating that free 70S ribosomes are real components of Bacillus stearothermophilus and Escherichia coli in exponential and stationary phases of growth.After pulses of radioactive leucine or uracil were given, protein synthesis was instantaneously stopped by chloramphenicol, or polypeptide growth was allowed to proceed to completion in slowly cooled cells. Analysis of label distribution in sucrose gradients showed that 70S monomers, and not subunits, are final products of translation.A ribosome cycle for protein synthesis in bacteria is proposed.A few years ago, it was proposed that in growing Escherichia coli, the polyribosomes are in equilibrium with a pool of ribosomal subunits. 1-3 Mangiarotti and Schlessinger did not find free 70S monomers in their extracts and therefore concluded that the 70S particles observed by other investigators came from the artificial breakdown of polysomes. However, several groups of workers,4-6 using methods gentle enough to preserve polyribosomal integrity, always observed 70S monomers in lysates of growing E. coli. Moreover, the 70S ribosomes did not bear nascent polypeptides, as could be shown in extracts from growing bacteria prepared immediately after a pulse of radioactive amino acids.4' These results suggest that most of the observed monomers are not an artifact produced by nuclease degradation of polyribosomes.In our studies on protein synthesis in Bacillus stearothermophilus7 (and unpublished results), we have repeatedly observed a 70S ribosome peak in bacterial lysates analyzed by sucrose density gradient centrifugation. When the cells from exponential growth phase were cooled rapidly or treated with chloramphenicol, the monomer peak amounted to 10-15 per cent of the total ribosomes. This percentage markedly increased either in exponentially growing cells harvested with slow cooling, or in bacteria at the stationary phase. In these cases the common feature is presumably the accumulation of the products of translation ending.Recently, Oppenheim et al.8 have obtained similar results with Azotobacter vinelandii disrupted very gently by direct osmotic shock.In this report we shall present evidence that in both B. stearothermophilus and E. coli the 70S monomers are liberated from the polyribosome upon termination of the reading of the messenger. These free 70S ribosomes no longer carry peptides and have lost the mRNA to which they were bound in polyribosome complexes. Before these monomers can participate in a new round of translation, they must be split into subunits. This transformation seems to occur through the action of a dissociating factor, as may be inferred from some of our preliminary results.

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“…For example, the number of ribosomal subunits in Landschutz ascites cells remains constant during dramatic changes in the relative proportions of monomers and polyribosomes in response to changes in the availability of amino acids (Hogan & Korner, 1968). The concentration of ribosomal subunits also remains constant in Azotobacter vinelandii during exponential growth and during the shift to stationary phase (Oppenheim, Scheinkbuks, Biava & Marcus, 1968).…”

Section: Co N4mentioning

confidence: 94%

Properties of the inactive ribosomal components in rat liver and hepatoma

Webb

Morris

1969

Biochemical Journal

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1. The relative concentrations of the inactive ribosomal components were compared in normal and regenerating rat liver and in two transplantable rat hepatomas (hepatomas 7800 and 5123D). 2. The size of the ribosomal-subunit pools in normal liver was not significantly affected by partial hepatectomy or neoplasia although, as shown previously, significant changes do occur in the monomer pool. 3. Further, the subunit pools in both liver and hepatoma were not significantly influenced by several treatments that caused dramatic changes in the size of the ribosomal monomer (and dimer) pools. 4. The high concentration of inactive monomers and dimers in the hepatomas appears to arise from limitations at the translational level, since they can be incorporated into pre-existing polyribosomes under the influence of cycloheximide.

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Polyribosomes in Azotobacter vinelandii

Cited by 27 publications

References 20 publications

Properties of initiation-free polysomes of Escherichia coli

Properties of initiation-free polysomes of Escherichia coli

PHYSIOLOGICAL ROLE OF 70 S RIBOSOMES IN BACTERIA

Properties of the inactive ribosomal components in rat liver and hepatoma

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