Structural dynamics of bacterial ribosomes
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Previous studies have shown that E. coli ribosomes may occur in several states that differ with respect to activity in protein synthesis and strength of subunit association. Both subunits may exist in the native a-state or one of the two denatured states b, c. For convenience small and capital letters are used to designate in couples the states of the small and large subunits respecitvely. Tight couples (aA) are stable on sucrose gradients at 6 mM Mg2+ and represent the most active states, whereas loose couples (aB) require 15 mM Mg2+ for stability. In the c-state neither subunit is capable of association with any partner. In the b-form 30S subunits cannot form couples; however, they can be reconverted to the a-form by thermal activation.
In the cell all ribosomes are in the a-states, which were preserved during cell breakage and purification at the very high Mg2+ concentration chosen (50 mM).
Denaturation of the native a subunits to the less active b and c forms is dependent on the ionic environment and temperature: the denaturation rate of 50S-a subunits increases by four orders of magnitude as the Mg2+ concentration is reduced from 10 mM to 0.1 mM. At 1 mM Mg2+ 50% denaturation requires 2 min at 37°C, 15 min at 0°C.
Exposure of ribosomes to hydrostatic pressures in excess of a critical pressure of ca. 1000 at (at 6 mM Mg2+) in a pressure chamber causes denaturation of the a-forms of the subunits into the characteristic b and c forms. The critical pressure increases and the rate of denaturation decreases with increasing Mg2+ concentration.
The electrophoretic mobility of ribosome subunits decreases by a factor 3.5 as the Mg2+ concentration is raised from 0.1 to 10 mM (at 50 mM NH4+) and by 20% as the NH4+ concentration is raised from 50 mM to 400 mM (at 3 mM Mg2+). This dependence of the ζ potential on the concentration of these ions supports the interpretation that Mg2+ and to a lesser extent NH4+ stabilize the tertiary structure of the ribsosomes primarily by reducing the repulsion between the negative phosphate groups in the RNA backbone.
These findings strongly argue that the molecular mechanisms of denaturation involve conformational rather than chemical changes, in agreement with the lack of evidence for chemical changes reported else-where.
Since the antagonism between Mg2+ and NH4+ in subunit association is not paralleled by antagonisms in charge compensation or denaturation, Mg2+ may have an additional function in association, such as the formation of specific Mg2+ bridges by coordination bonds within or between subunits.
The rapid rate of denaturation at low Mg2+ concentrations complicates measurements of the association constant of the equilibrium between subunits and couples and accounts for the hysteresis of the dissociation/association curves reported in the literature.
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- Debye, P., Hui Bon Hoa, G., Douzou, P., Godefroy-Colburn, T., Graffe, M., Grunberg-Manago, M.: Ribosomal subunit interaction as studied by light scattering. Evidence of different classes of ribosome preparations. Biochemistry (Wash.) 14, 1553–1558 (1975)Google Scholar
- Diggelen, O.P. van, Oostrom, H., Bosch, L.: The association of ribosomal subunits of Escherichia coli. 2. Two types of association products differing in sensitivity to hydrostatic pressure generated during centrifugation. Europ. J. Biochem. 39, 511–523 (1973)Google Scholar
- Duin, J. Ban, Dieijen, G. van, Knippenberg, P. van, Bosch, L.: Different species of 70S ribosomes of Escherichia coli and their dissociation into subunits. Europ. J. Biochem. 17, 433–440 (1970)Google Scholar
- Gavrilova, L.P., Ivanov, D.A., Spirin, A.S.: Studies on the structure of ribosomes; III. Stepwise unfolding of the 50S particles without loss of ribosomal protein. J. molec. Biol. 16, 473–489 (1966)Google Scholar
- Gesteland, R.F.: Unfolding of Escherichia coli ribosomes by removal of magnesium. J. molec. Biol. 18, 356–371 (1966)Google Scholar
- Ginzbung, I., Miskin, R. Zamir, A.: N-ethyl maleimide as a probe for the study of functional sites and conformations of 30S ribosomal subunits. J. molec. Biol. 79, 481–494 (1973)Google Scholar
- Hapke, B., Noll, H.: Structural dynamics of bacterial ribosomes. IV. Classification of ribosomes by subunit interaction. J. molec. Biol. 105, 97–109 (1976)Google Scholar
- Infante, A.A., Baierlein, R.: Pressure-induced dissociation of sedi menting ribosomes: effect on sedimentation patterns. Proc. nat. Acad. Sci. (Wash.) 68, 1780–1785 (1971)Google Scholar
- Kikuchi, A., Monier, R.: Association of subunits in purified preparations of Escherichia coli ribosomes. FEBS Letters 11, 157–162 (1970)Google Scholar
- Nierhaus, K.H., Dohme, F.: Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc. nat. Acad. Sci. (Wash.) 71, 4713–4717 (1974)Google Scholar
- Noll, H.: Konstruktion und Wirkungsweise neuer Instrumente zur Erzielung höchster Auflösung bei der Sedimentationsanalyse in Dichtegradienten und ihre Anwendung zum Studium der Strukturdynamik von Ribosomen. Vjschr. Naturforsch. Ges. Zürich 116, 377–402 (1971)Google Scholar
- Noll, H., Noll, M., Hapke, B., Dieijen, G. van: The mechanism of subunit interaction as a key to the understanding of ribosome function. In: Regulation of transcription and translation in eukaryotes, Mosbach Colloquium No. 24 (E.K.F. Bautz, P. Karlson and H. Kersten, eds.), pp. 257–311 New York: Springer 1973cGoogle Scholar
- Noll, M.: Chain initiation, elongation, and termination of the coat protein hexapeptide of R 17 amB2 in a completely purified translation system from Escherichia coli. Thesis, Northwestern University (1972)Google Scholar
- Noll, M., Hapke, B., Noll, H.: Structural dynamics of bacterial ribosomes. II. Preparation and characterization of ribosomes and subunits active in the translation of natural messenger RNA. J. molec. Biol. 80, 519–529 (1973b)Google Scholar
- Noll, M., Hapke, B., Schreier, M., Noll, H.: Structural dynamics of bacterial ribosomes. I. Characterization of vacant couples and their relation to complexed ribosomes. J. molec. Biol. 75, 281–294 (1973a)Google Scholar
- Noll, M., Noll, H.: Translation of R 17 RNA by Escherichia col ribosomes; initiator transfer RNA-directed binding of 30S subunits to the starting codon of the coat protein gene. J. molec. Biol. 89, 477–494 (1974a)Google Scholar
- Noll, M., Noll, H.: Structural synamics of bacterial ribosomes. III. Quantitative conversion of vacant ribosome couples into an initiation complex with R 17 RNA as messenger. J. molec. Biol. 90, 237–251 (1974b)Google Scholar
- Noll, M., Noll, H.: Structural dynamics of bacterial ribosomes. V. Magnesium dependent equilibrium of tight couples with subunits: measurement of dissociation constants and exchange rates. J. molec. Biol. 105, 111–130 (1976)Google Scholar
- Spirin, A.S.: Some problems concerning the macromolecular structure of ribonucleic acids. Progr. Nucl. Acid Res. 1, 301–345 (1963)Google Scholar
- Spirin, A.S., Sabo, B., Kovalenko, V.A.: Dependence of dissociation-association of uncharged ribosomes of Escherichia coli on the Mg2+ concentration, ionic strength, pH and temperature. FEBS letters 15, 197–200 (1971)Google Scholar
- Stahli, C.: Conformational instability as the major cause of inactiviation of prokaryotic ribosomes. Thesis, Northwestern University (1975)Google Scholar
- Stahli, C., Noll, H.: Manuscript in preparation.Google Scholar
- Talens, A., Diggelen, O.P. van, Brongers, M., Popa, L.M., Bosch, L.: Electrophoretic separation of Escherichia coli ribosomal particles on polyacrylamide gels. Europ. J. Biochem. 37, 121–133 (1973)Google Scholar
- Traub, P., Nomura, M.: Structure and function of E. coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc. nat. Acad. Sci. (Wash.) 59, 777–784 (1968)Google Scholar
- Walters, J.A.L.I., Os, G.A.J. van: The dissociation and association behavior of yeast ribosomes. Biochim. biophys. Acta (Amst.) 199, 453–463 (1970)Google Scholar
- Wishnia, A., Boussert, A., Graffe, M., Dessen, P.H., Grunberg-Manago, M.: Kinetics of the reversible association of ribosomal subunits: stopped-flow studies of the rate law and of the effect of Mg2+. J. molec. Biol. 93, 499–516 (1975)Google Scholar
- Zamir, A., Miskin, R., Elson, D.: Thactivation and reactivation of ribosomal subunits: amino acyl transfer RNA binding of the 30S subunit of Escherichia coli. J. molec. Biol. 60, 347–364 (1971)Google Scholar
- Zitomer, R.S., Flaks, J.G.: Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association. J. molec. Biol. 71, 263–279 (1972)Google Scholar