Effects of Ribonuclease T\ on Escherichia coli Ribosomes

Purified Escherichia coli ribosomes were digested with a low concentration of a water-insoluble derivative of RNase Ti. Such a treatment resulted in an impairment of activity of these ribosomes to support poly(U)-directed [14C]polyphenylalanine synthesis. The AA-tRNA and poly-(U) binding activities were also reduced. The decrease in these activities was proportional to the duration of the enzymatic treatment. The kinetics of inactivation of these ribo-somal particles were biphasic. The initial rate of inactivation was rapid followed by a slower rate, about 22 % of the initial rate. The rate of inactivation of a mixture of 30S and 50S subunits was faster than that of the 70S particles. The RNAs remaining in the ribosomal particles after such an enzymatic treatment was analyzed by column chromatography on DEAE-cellulose at pH 6.5 and 2.7. Relatively few fragments were observed. The 3′ -terminal oligonucleotides of the rRNAs which had been labeled with tritiated NaBH₄ were found among these protected RNA regions. Uridine was found to be the major terminus of these RNA fragments.