STABILIZATION OF A RIBOSOMAL-RNA TERTIARY STRUCTURE BY RIBOSOMAL-PROTEIN L11

Interactions between ribosomal protein L11 and a domain of large subun it rRNA have been highly conserved and are essential for efficient pro tein synthesis. To study the effects of L11 on rRNA folding, a homolog of the Escherichia coli L11 gene has been amplified from Bacillus ste arothermophilus DNA and cloned into a phage T7 polymerase-based expres sion system. The expressed protein is 93% homologous to the L11 homolo g from Bacillus subtilis, denatures at temperatures above 72 degrees C , and has nearly identical rRNA binding properties as the Escherichia coli L11 in terms of RNA affinity constants and their dependences on t emperature, Mg2+ concentration, monovalent cation, and RNA mutations. Mg2+ and NH4+ are specifically bound by the RNA-protein complex, with apparent ion-RNA affinities of 1.6 mM(-1) and 19 M(-1), respectively, at 0 degrees C. The effect of the thermostable L11 on the unfolding of a 60 nucleotide rRNA fragment containing its binding domain has been examined in melting experiments. The lowest temperature RNA transition , which is attributed to tertiary structure unfolding, is stabilized b y similar to 25 degrees C, and the interaction has an intrinsic enthal py of similar to 13 kcal/mol. The thermal stability of the protein-RNA complex is enhanced by increasing Mg2+ concentration and by NH4+ rela tive to Na+. Thus L11, NH4+, and Mg2+ all bind and stabilize the same rRNA tertiary interactions, which are conserved and presumably importa nt for ribosome function.