PSEUDOKNOT IN DOMAIN-II OF 23-S RIBOSOMAL-RNA IS ESSENTIAL FOR RIBOSOME FUNCTION

The structure of domain II in all 23 S (and 23 S-like) rRNAs is constr ained by a pseudoknot formed between nucleotides 1005 and 1138, and be tween 1006 and 1137 (Escherichia coli numbering). These nucletoides ar e exclusively conserved as 1005C . 1138G and 1006C . 1137G pairs in al l Bacteria, Archaea and chloroplasts, whereas 1005G . 1138G and 1006U . 1137A pairs occur in Eukarya. We have mutagenised nucleotides 1005C --> G, 1006C --> U, 1137G --> A and 1138G --> C, both individually and in combinations, in a 23 S rRNA gene from the bacterium E. coli. The ability of 23 S rRNA to support cell growth is reduced when either of these base-pairs is disrupted, and it is completely abolished upon dis ruption of both base-pairs. Each mutant 23 S rRNA is assembled into 50 S subunits, but the mutant subunits do not stably interact with 30 S to engage in protein synthesis. Enzymatic and chemical probing of ribo somal particles reveals increased accessibility in the rRNA structure close to the sites of the mutations. The degree to which the mutations increase rRNA accessibility correlates with the severity of their phe notypic effects. Nucleotide 1131G is extremely reactive to dimethyl su lphate modification in wild-type subunits and ribosomes, but is render ed unreactive when either the pseudoknot is broken or when the r-prote ins are removed. The structure of the pseudoknot region is possibly in fluenced by interaction of an r-protein at or close to the pseudoknot. Re-establishing the pseudoknot Watson-Crick interactions with one ''e ukaryal'' (1005G . 1138C or 1006U 1137A) pair and one ''bacterial'' C G pair largely restores the structure and function of the rRNA. Bacter ial ribosomes containing both these eukaryal pairs also participate in protein synthesis, although at much reduced efficiency, and the struc ture of their pseudoknot region is partially open and accessible.