Inactivation and reactivation of ribosomal subunits: The peptidyl transferase activity of the 50 s subunit of Escherichia coli

Miskin, Ruth; Zamir, Ada; Elson, David

doi:10.1016/0022-2836(70)90435-3

Cited by 124 publications

(45 citation statements)

References 29 publications

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“…A similar effect of NH,+ in vivo was observed by Rigano 8z Violante (I 973) during derepression of nitrate reductase synthesis by Cyanidium caldarium. Ammonium ions stimulate cell-free amino acid incorporation (Lubin & Ennis, 1964) ; high NH$ concentrations are required for ribosomal stability in vitro (Hartman, Nolan & Amaya, 1971) and for the peptidyl transferase reaction (Miskin, Zamir & Elson, 1970). Under argon, only a limited amount of nitrogenase was derepressed, as previously observed by Parejko & Wilson, 1970; Casamino acids added prior to derepression increased the final activity obtained in one instance.…”

Section: Discussionmentioning

confidence: 71%

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

Tubb

Postgate

1973

Journal of General Microbiology

103

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SUMMARYSulphate-limited continuous cultures of Klebsiella pneumoniae showed a proportional repression of nitrogenase activity with increasing concentrations of ammonium ion in the influent medium. A fully repressed population had a 50 % greater bacterial density than a fully derepressed one. On derepression, synthesis of nitrogenase lagged for 90 min after exhaustion of NH,+ from the medium but was complete within one doubling time. Casamino acids did not decrease the prefixation lag obtained under sulphate-limited conditions in the chemostat. Longer lags were obtained when NH,+ was exhausted under N-limited conditions. Such lags were decreased by Casamino acids, yeast extract, or L-aspartate. Aspartate-N completely repressed nitrogenase under sulphate-or carbon-limited conditions but not under nitrogen limitation. In the initial stages of repression of nitrogenase synthesis by NH,+, apparent production of active enzyme continued for a short time in the absence of protein synthesis de novo; nitrogenase was then diluted out as the organisms multiplied. Repression by NH,+ was at the level of mRNA transcription according to studies with rifampicin and chloramphenicol. ' Coding capacity' for nitrogenase synthesis declined with a half-life of about 4.5 min following inhibition of RNA synthesis with rifampicin. NH,+ did not influence the decay rate but stimulated translation of such nitrogenase-specifying mRNA as had been initiated.

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Section: Discussionmentioning

confidence: 71%

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

Tubb

Postgate

1973

Journal of General Microbiology

103

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“…Potassium seems to be required for at least two essential ribosomal reactions. First, K + ions are needed for the peptidyl transferase center to assume its functional conformation (42). Second, our sequence and structure comparisons indicate that the key translation factors are K + -dependent GTPases (SI Appendix, Figs.…”

Section: Resultsmentioning

confidence: 97%

Origin of first cells at terrestrial, anoxic geothermal fields

Mulkidjanian

Bychkov²,

Dibrova³

et al. 2012

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

375

336

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All cells contain much more potassium, phosphate, and transition metals than modern (or reconstructed primeval) oceans, lakes, or rivers. Cells maintain ion gradients by using sophisticated, energydependent membrane enzymes (membrane pumps) that are embedded in elaborate ion-tight membranes. The first cells could possess neither ion-tight membranes nor membrane pumps, so the concentrations of small inorganic molecules and ions within protocells and in their environment would equilibrate. Hence, the ion composition of modern cells might reflect the inorganic ion composition of the habitats of protocells. We attempted to reconstruct the "hatcheries" of the first cells by combining geochemical analysis with phylogenomic scrutiny of the inorganic ion requirements of universal components of modern cells. These ubiquitous, and by inference primordial, proteins and functional systems show affinity to and functional requirement for K + , Zn 2+ , Mn 2+, and phosphate. Thus, protocells must have evolved in habitats with a high K + /Na + ratio and relatively high concentrations of Zn, Mn, and phosphorous compounds. Geochemical reconstruction shows that the ionic composition conducive to the origin of cells could not have existed in marine settings but is compatible with emissions of vapor-dominated zones of inland geothermal systems. Under the anoxic, CO 2 -dominated primordial atmosphere, the chemistry of basins at geothermal fields would resemble the internal milieu of modern cells. The precellular stages of evolution might have transpired in shallow ponds of condensed and cooled geothermal vapor that were lined with porous silicate minerals mixed with metal sulfides and enriched in K + , Zn 2+, and phosphorous compounds.

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“…After 10 min at 0WC, the reaction was terminated with 10 Mul of 10 N KOH and samples were incubated for 10 min at 37°C to hydrolyze fMet-methyl ester, which is formed in this assay (27,29). 1 ml of 1 M sodium phosphate buffer (pH 7.0) was then added and the product was extracted with ethyl acetate.…”

Section: Methodsmentioning

confidence: 99%

Role of 5S RNA in the Functions of 50S Ribosomal Subunits

Erdmann

Fahnestock

Higo

et al. 1971

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

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ABSTRACT50S ribosomal subunits from Bacillus stearothermophilus can be reconstituted from their dissociated components, namely a 5S RNA-free protein fraction, a 5S RNA-free 23S ribosomal RNA fraction, and purified 5S RNA. The biological activity of reconstituted particles in polypeptide synthesis is dependent on the presence of 5S RNA. In the absence of 5S RNA, particles are produced that have greatly reduced activity in (a) polypeptide synthesis directed by synthetic, as well as natural, messenger RNA, (b) peptidyl transferase assay, (c Since 5S RNA was discovered as a component of the large ribosomal subunit (1), many studies have been done on its structure and biosynthesis. The primary sequence of 5S RNA from Escherichia coli was first elucidated by Brownlee, Sanger, and Barell (2). Yet, the function of 5S RNA has remained unknown. Several workers devised methods to remove 5S RNA from 50S subunits and found that removal of 5S RNA led to inactivation of 50S subunits (3-5). However, these workers were unable to restore activity to these inactive particles by adding 5S RNA. Thus, it was not possible to decide whether the inactivation was due to the removal of 5S RNA or to some structural disorganization of the particles caused by the treatment used.We have recently succeeded in reconstituting 50S ribosomal subunits from Bacillus stearothermophilus from their dissociated molecular components (6). Using this reconstitution system, we have now shown that 5S RNA is essential for the overall activity of 50S subunits in polypeptide synthesis. This paper describes information on the function of 5S RNA obtained in this system. MATERIALS AND METHODSThe following buffers are used: TMA-I; 0.01 M Tris HCl (pH 7.4)-0.01 M MgC12-0.03 M NH4Cl-6 mM 2-mercaptoethanol. TMA-II; Same as TMA-I, except 0.3 mM MgCl2. TRI; 0.03 M Tris * HC1 (pH 7.4)-0.02 M MgCl2-1 M KC1-6 mM 2-mercaptoethanol. TRO B. stearothermophilus 799 was grown at 610C to early-log phase in medium L (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 1 g of glucose per liter of H20). The pH is adjusted to 7.0 with NaOH. Cells were ground with two times their weight of alumina at 00C and extracted with five times the cell weight of TMA-I buffer containing 2 Ag/ml of RNase-free DNase. Alumina, unbroken cells, and cell debris were removed by centrifugation, first at 20,000 X g for 20 min, and then 78,000 X g for 30 min, to give the crude extract. Crude 70S ribosomes were obtained by centrifugation of the extract at 78,000 X g for 10 hr. The 70S ribosomes were then dialyzed against TMA-II overnight at 40C and the dissociated ribosomal subunits were separated and purified (7). All ribosomes were stored in TMA-I at -80'C.50S subunits (900 A260/ml in TMA-I) were treated with an equal volume of 8 M urea-4 M LiCl solution at 00C for 48 hr, and then centrifuged at low speed. The RNA pellet was washed once at 0C with a mixture (1:1) of TMA-I and 8 M urea-A M LiCl solution and then dissolved in water ("RNA 50" fraction). This fraction contains 23S RNA with one protein (...

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Inactivation and reactivation of ribosomal subunits: The peptidyl transferase activity of the 50 s subunit of Escherichia coli

Cited by 124 publications

References 29 publications

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

Control of Nitrogenase Synthesis in Klebsiella pneumoniae

Origin of first cells at terrestrial, anoxic geothermal fields

Role of 5S RNA in the Functions of 50S Ribosomal Subunits

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