Conformational Studies ofEscherichia coli Ribosomes with the Use of Acridine Orange as a Probe

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Abstract
The interaction of E. coli vacant ribosomes with acridine orange (AO) was studied, to obtain conformational information about rRNA in ribosomes. Acridine orange binds to an RNA in 2 different modes: cooperative outside binding with stacking of bound AO''s and intercalation between nucleotide bases. Free 16S and 23S rRNA have almost identical affinities to AO. At 1 mM Mg2+, AO can achieve stacking binding on .apprx. 40% of rRNA phosphate groups. The number of stacking binding sites falls to .apprx. 1/3 in the 30S subunit in comparison with free 16S rRNA. In the 50S subunit the number of stacking binding sites is 1/5 in comparison with free 23S rRNA. Mg2+ ions are more inhibitory for the binding of AO to ribosomes than to free rRNA. The strength of stacking binding appears to be more markedly reduced by Mg2+ in active ribosomes than in rRNA. Tight couple 70S particles are less accessible for stacking binding than free subunits. The 30S subunits that have irreversibly lost the capability for 70S formation under low Mg2+ conditions have an affinity to AO that differs from that of active 30S but is similar to that of free rRNA, although the number of stacking binding sites is little changed by the inactivation. Ribosomes (70S and 30S) with stacking bound AO''s have normal sedimentation constants, but the 50S subunits reversibly form aggregates.

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