Effects of antisense DNA against the α-sarcin stem-loop structure of the ribosomal 23S rRNA

Abstract

Antisense DNAs complementary against various sequences of the or-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S 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 α-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of α-sarcin. The RNase α-sarcin seems to approach its cleavage site from the 3' half of the loop of the α-sarcin domain. Hybridization is efficiently achieved at 37°C and can extend at least into the 3' strand of the stem of the α-sarcin domain. However, the inhibition of α-sarcin activity is observed at 30°C but not at 37°C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25°C. The results imply that the α-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the α-sarcin domain.