Higher order structure in the 3'-minor domain of small subunit ribosomal RNAs from a gram negative bacterium, a gram positive bacterium and a eukaryote.

An experimental approach was used to determine and compare the highest order structure within the 150 to 200 nucleotides at the 3'-ends of the RNAs from the small ribosomal subunits of Escherichia coli, Bacillus stearothermophilus and Saccharomyces cerevisiae. Chemical reagents were employed to establish the degree of stacking and/or accessibility of each adenosine, guanosine and cytidine. The double helices were probed with a cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were localized with the single strand-specific ribonucleases A, T1, T2 and S1. The data enabled the various minimal secondary structural models, proposed for the 3'-regions of the E. coli and S. cerevisiae RNAs, to be critically examined, and to demonstrate that the main common features of these models are correct. The results also reveal the presence and position of additional higher order structure in the renatured free RNA. It can be concluded that a high level of conservation of higher order structure has occurred during the evolution of the gram negative and gram positive eubacteria and the eukaryote in both the double helical regions and the "unstructured" regions. Several unusual structural features were detected. Multiple G X A pairings in two of the putative helices, which are compatible with phylogenetic sequence comparisons, are strongly supported by the occurrence of cobra venom ribonuclease cuts adjacent to, and in one case between, these pairings. Evidence is also provided for the stacking of an A X A pair within a double helix of the yeast RNA. Other special structural features include adenosines bulged out from double helices; such nucleotides, which are hyper-reactive, have been implicated in protein recognition in 5 S ribosomal RNA. The 3'-terminal regions of the RNAs are particularly important for the functioning of the ribosome. They are involved in mRNA, tRNA and ribosomal factor binding. The results reveal that while the functionally important RNA sequences tend to be conserved, they are not always accessible in the free RNA; the pyrimidine-rich "Shine and Dalgarno" sequence, for example, which is involved in mRNA recognition, occurs in a double helix in both eubacterial RNAs.