EFFECTS OF ANTISENSE DNA AGAINST THE ALPHA-SARCIN STEM-LOOP STRUCTUREOF THE RIBOSOMAL 23S RIBOSOMAL-RNA

Antisense DNAs complementary against various sequences of the alpha-sa rcin domain (C2640-G2674) of 235 rRNA from Escherichia coli were hybri dized to naked 235 rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the r esulting fragments and blockage of the action of alpha-sarcin. The RNa se alpha-sarcin seems to approach its cleavage site from the 3' half o f the loop of the alpha-sarcin domain. Hybridization is efficiently ac hieved at 37 degrees C and can extend at least into the 3' strand of t he stem of the alpha-sarcin domain. However, the inhibition of alpha-s arcin activity is observed at 30 degrees C but not at 37 degrees C. Fo r a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha -sarcin domain.