Stabilization of a ribosomal RNA tertiary structure by ribosomal protein L11

Abstract

Interactions between ribosomal protein L11 and a domain of large subunit rRNA have been highly conserved and are essential for efficient protein synthesis. To study the effects of L11 on rRNA folding, a homolog of the Escherichia coli L11 gene has been amplified from Bacillus stearothermophilus DNA and cloned into a phage T7 polymerase-based expression system. The expressed protein is 93% homologous to the L11 homolog from Bacillus subtilis, denatures at temperatures above 72°C, and has nearly identical rRNA binding properties as the Escherichia coli L11 in terms of RNA affinity constants and their dependences on temperature, Mg2+ concentration, monovalent cation, and RNA mutations. Mg2+ and NH+4 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°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 by ∼25°C, and the interaction has an intrinsic enthalpy of ∼13 kcal/mol. The thermal stability of the protein-RNA complex is enhanced by increasing Mg2+ concentration and by NH+4 relative to Na+. Thus L11, NH+4, and Mg2+ all bind and stabilize the same rRNA tertiary interactions, which are conserved and presumably important for ribosome function. © 1995 Academic Press Limited.