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.
A study of the nature of these states and their transitions gave the following results.
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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- Structural dynamics of bacterial ribosomes
Molecular and General Genetics MGG
Volume 153, Issue 2 , pp 159-168
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