Magnesium-dependent interaction of 30S ribosomal subunits with antibodies to N6, N6-dimethyladenosine

Abstract

The modified nucleoside N6, N6-dimethyladenosine occurs in Escherichia coli 16S ribosomal RNA only in two successive positions near its 3' end. Antibodies directed against dimethyladenosine were induced with a nucleoside-albumin conjugate. As measured by second antibody precipitation of immune complexes, antidimethyladenosine antibodies bound 30S ribosomal subunits, ribosomal core particles, and ribosomal RNA which contain dimethyladenosine but showed little cross-reactivity with RNA or ribosomal subunits from a kasugamycin-resistant mutant which lacks dimethyladenosine. Antibody binding to ribosomal subunits was strongly influenced by the concentration of magnesium ion in the reaction medium and by the prior treatment of the subunits. Functionally active 30S subunits showed a striking binding optimum at 2-4 mM Mg2+; this optimum disappeared if the subunits were inactivated by dialysis against low concentrations of magnesium ion. Instead, the inactivated subunits showed a gradual increase in antibody binding as the magnesium ion concentration was raised to 20 mM; binding of 16S ribosomal RNA or subribosomal core particles from 30S subunits gave qualitatively similar curves, with no evidence of a low [Mg2+] optimum. The stability of antibody-subunit complexes was also found to depend upon subunit conformation and magnesium ion concentration; the half-life of an inactivated subunit-antibody complex (15 mM Mg2+) averaged 130 min, while active subunit-antibody complexes (3 mM Mg2+) had an average half-life of 70 min. More of the immune complexes with inactivated subunits were found to survive sucrose gradient sedimentation (relative to active subunits), and the concentration of subunits needed to halve antibody binding of [3H]-N6, N6-dimethyladenosine was lower with inactivated subunits. The results suggest that the antibody binding optimum seen with active subunits at 2-4 mM Mg2+ represents a dynamic aspect of the three-dimensional ribosomal subunit structure; a site near the 3' end of the RNA is involved, and both the availability of the modified nucleoside to an antibody probe and the stability of the resulting complexes are involved.