Salt response of ribosomes of a moderately halophilic bacterium

Wydro, Robert; Kogut, Margot; Kushner, D. J.

doi:10.1016/0014-5793(75)80453-4

Cited by 13 publications

(11 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For locating the 70S, 50S, and 30S ribosomal particles, parallel experiments were carried out in which purified ribosomes were centrifuged in appropriate gradients in the presence of 18 or 1 mM Mg2+; with 1 mM Mg2+, the 70S ribosomes dissociated into 50S and 30S subunits (18). (Note that we are using 70S, 50S, and 30S to indicate the usual forms of ribosomes, though the actual "S" values determined for V. costicola were slightly different: 64S, 48S, and 28S [18].)…”

Section: Methodsmentioning

confidence: 99%

“…(Note that we are using 70S, 50S, and 30S to indicate the usual forms of ribosomes, though the actual "S" values determined for V. costicola were slightly different: 64S, 48S, and 28S [18].) RESULTS Importance of different parts of the protein-synthesizing system.…”

Section: Methodsmentioning

confidence: 99%

“…Although the sedimentation behavior of the ribosomes does not change at high Cl-ion concentrations (18), some ribosomal proteins may still be lost (R. M. Wydro, Ph.D. thesis, University of Ottawa, Ottawa, Ontario, Canada, 1977). However, this loss did not damage the proteinsynthesizing ability of the ribosomes.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions

1989

View full text Add to dashboard Cite

In vitro protein synthesis in Vibrio costicola [poly(U)-directed incorporation of phenylalanine] was studied.The extent of protein synthesis was limited by the number of ribosomes present. Density gradient centrifugation experiments suggested that, after runoff of ribosomes from the artificial messenger, the 50S subunit was unable to attach to the 30S-messenger complex. As shown previously (M. Kamekura and D. J. Kushner, J. Bacteriol. 160:385-390, 1984), Cl-ions inhibited protein synthesis; indeed, the highest rate of synthesis took place in the lowest attainable Cl-concentration (37 mM). The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. The strongest reversal was seen when both glutamate and betaine were present. Cl-ions car! prevent binding of ribosomes to poly(U) and displace ribosomes already bound to this artificial messenger. The effects of Cl-ions on binding were also reversed by glutamate and betaine. Cl-ions did not affect accuracy of translation; they were shown previously (Kamekura and Kushner, J. Bacteriol. 160:385-390, 1984) not to affect phenylalanyl-tRNA synthetase. It was also found that washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. On the contrary, these ribosomes were more active than before they were washed. We conclude that the main site of action of Cl-in the system studied is on the binding of ribosomes to the mRNA.Vibrio costicola is a moderate halophile which grows in media containing a wide range (0.4 to 3.5 M) of NaCl concentrations, with its optimum growth occurring at a concentration of around 1 M NaCl (7). Although its cellassociated concentration of Na+ and K+ ions is generally equal to the external Na+ concentration (15), in vitro protein synthesis [poly(U)-dependent phenylalanine incorporation] by cellular extracts of this bacterium, after growth in the presence of 1 M NaCl, was found to have maximal activity in 0.1 to 0.2 M concentrations of NaCl, KCl, or NH4C1 and no, or very little, activity in 0.6 M concentrations of these salts. Amino acid incorporation dependent on "endogenous" mRNA had similar responses to these salts (19).The inhibitory effects were shown to be due mainly to the C1-ions, which are normally excluded from the cells: V. costicola grown in the presence of 1.0 M NaCl contains only 0.2 M C1-(6). The effects of higher concentrations of these ions were partly reversed by a number of organic substances, including glycine betaine (betaine) and glutamate, which are accumulated within the cells.The site of action of C1-has not yet been identified. It is not phenylalanyl-tRNA synthetase, which is barely inhibited by as much as 0.9 M NaCl (6). In this paper, we report further studies of the in vitro protein-synthesizing system of V. costicola, with special reference to the site of action of Cl-ions.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions

1989

View full text Add to dashboard Cite

show abstract

“…When chloride was replaced by other anions such as glutamate, sulfate, or acetate, high in vitro protein synthesis rates were found at salt concentrations as high as 0.6 M, both in Salinivibrio costicola and in Chromohalobacter canadensis (Choquet et al, 1989;Kamekura and Kushner, 1984;Kushner, 1989Kushner, , 1991Wydro et al, 1977). The ribosomes of Salinivibrio costicola showed an unusual sedimentation behavior in sucrose gradients: association of subunits to complete ribosomes took only place in high salt (Wydro et al, 1975).…”

Section: The Ribosomal Proteins Of Halophilic Archaeamentioning

confidence: 99%

Halophilic Microorganisms and their Environments

Oren

2002

Cellular Origin, Life in Extreme Habitats and Astrobiology

404

294

View full text Add to dashboard Cite

“…Those of V. costicola are stable in both high and low salt concentrations, while those of E. coli are stable only in low salt concentrations (37). Higher concentrations of NH4Cl are needed to remove initiation factors from the ribosomes of V. costicola than from those of E. coli.…”

mentioning

confidence: 99%

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola

Choquet

Kushner

1990

J Bacteriol

View full text Add to dashboard Cite

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichwa coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNAmet (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA4et showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4' (as glutamate). Higher salt concentrations, especially those with Cl-as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl-ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl-ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4C1, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl-ions inhibited the attachment of initiation factors to tRNAmet but had little effect on binding of initiation factors to R17 RNA.Polyuridylic acid [poly(U)]-directed incorporation of phenylalanine by cell extracts of the moderate halophile Vibrio costicola is inhibited by Cl-ions (9), which prevent ribosome binding to the artificial mRNA (3).There are a number of differences between a translation system directed by poly(U) and that directed by a natural mRNA. Poly(U) has no Shine-Dalgarno sequence, which normally plays an important role in the initial attachment of natural mRNAs to ribosomes. At MATERIALS AND METHODS Bacterial culture. V. costicola NRC 37001 was grown and harvested as described previously (9).Preparation of cellular extracts, ribosomes, and initiation factors. S-30 extract, that is, the cellular material remaining in the supernatant after centrifuging for 30 min at 30,000 x gmax (Beckman type 42.1 rotor), was prepared as described previously (9).The S-150 extract and the low-salt-washed ribosomes were obtained by centrifuging the S-30 extract at 150,000 x gmax for 3 h at 4°C. The soluble S-150 fraction and the ribosomal pellet were then dialyzed or washed and stored according to the procedure of Kamekura and Kushner (9).A different low-salt extraction buffer than the one described by Kamekura and Kushner (9) was used in the preparation of the cellular extracts and of the ribosomes. It contained 124 mM ammonium glutamate, 8 mM magnesium ace...

show abstract

Salt response of ribosomes of a moderately halophilic bacterium

Cited by 13 publications

References 11 publications

In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions

In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions

Halophilic Microorganisms and their Environments

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola

Contact Info

Product

Resources

About