152] Techniques for measuring specific sRNA binding to Escherichia coli ribosomes

Abstract

This chapter describes the techniques for measuring specific soluble RNA (sRNA) binding to Escherichia coli ribosomes. Specific sRNA binds to the template ribosome complex. The binding is specific, in which only the sRNA coded for in the messenger RNA (mRNA) sequence is stimulated to bind to the ribosomes. The bound sRNA sediments faster than the rest of sRNA during the sucrose density gradient centrifugation. The faster-sedimenting bound sRNA is assayed for its amino acid acceptor ability after the sucrose density gradient centrifugation. After the sucrose density gradient centrifugation of the mixture, the amounts of the various amino acid sRNA's in each fraction are determined by measuring the incorporation of 14C-amino acid into the fraction that is insoluble in cold trichloroacetic acid but soluble in hot trichloroacetic acid. The radioactive aminoacyl sRNA formed is soluble in hot trichloroacetic acid and insoluble in cold trichloroacetic acid. Radioactivity incorporated into the protein fraction is insoluble in both hot and cold trichloroacetie acid. It is, therefore, possible to measure the amount of each aminoacyl sRNA by subtracting the radioactivity insoluble in hot trichloroacetic acid from that insoluble in cold trichloroacetic acid. The sRNA sedimenting faster than the rest of sRNA is regarded as bound sRNA. Specific aminoacyl sRNA, like sRNA, binds to the template ribosome complex. Ribosomes are absorbed by Millipore filter quantitatively, while aminoaeyl sRNA is not. Using aminoacyl-14C sRNA, the ribosome-bound aminoacyl-14C sRNA can be conveniently measured by counting the radioactivity retained on the Millipore filter.