Structural analysis of prokaryotic and eukaryotic 5S rRNAs by RNase H

Abstract

The availabilities of single-stranded 5S rRNA regions c, d and d′ for base pairing interactions were analyzed by using synthetic DNA oligomers. Hybrid formation was detected by the endonucleolytical mode of the RNA-DNA specific action of RNase H. Provided that the hybrid interaction involved 6 successive base pairs, 5S rRNA loop c nucleotides 42–47 displayed accessibility in Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus 5S rRNAs as well as in eukaryotic 5S rRNAs from Sacharomyces carlsbergensis, Rattus rattus and Equisetum arvense. Investigating eubacterial 5S rRNA regions d and d′ (nucleotides 71–76 and 99–105, respectively), susceptibility was observed in E. coli 5S rRNA which, however, decreases in B. stearothermophilus and even more so in T. thermophilus 5S rRNA. For additional evaluation of the data obtained by RNase H cleavage, association constants of the hexanucleotides were determined by equilibrium dialysis at 4°C for B. stearothermophilus 5S rRNA. The results obtained reveal that nucleotides 36–41 of B. stearothermophilus 5S rRNA are inaccessible for Watson-Crick interaction, which suggests that this part of loop c is in a structurally constrained configuration, or buried in the tertiary structure or involved in tertiary interactions.