FIGURE 4.

Interaction between RbgA and ribosome in presence of different guanine nucleotides. Binding of RbgA to purified ribosomal subunits was tested by an in vitro binding assay in which 60 pmol of RbgA was preincubated with 1.5 mm GDP, GTP, GMPPNP, or pppGpp before the addition of 10 pmol of purified 45 S or 50 S ribosomal subunits. The mixtures were incubated for an additional 15 min at 37 °C, and free RbgA was filtered off by centrifugation through a 100-kDa cutoff Microcon column. The columns were washed three times, first with buffer B and then twice with buffer C (high salt buffer). RbgA-ribosome complexes were eluted, and bound RbgA was detected by immunoblotting using anti-RbgA antibody.