Protein-RNA Dynamics in the Central Junction Control 30S Ribosome Assembly

Abstract

Interactions between ribosomal proteins and ribosomal RNA (rRNA) facilitate the formation of functional ribosomes. S15 is a central domain primary binding protein that has been shown to trigger a cascade of conformational changes in 16S rRNA, which result in the functional structure of the central domain. Previous biochemical and structural studies in vitro have revealed that S15 binds a three-way junction of helices 20, 21 and 22, including nucleotides 652-654 and 752-754. All junction nucleotides except 653 are highly conserved in bacteria implying that these nucleotides are functionally important. To identify functionally important sequences and structural elements within the junction, nucleotides 652-654 and 752-754 were subjected to saturation mutagenesis and functional mutants were selected and analyzed. Only 74 mutants were isolated with greater than 4% ribosome function in vivo. To determine whether S15 binding was affected by mutations in the junction loop, S15 was cloned and over-expressed with the junction mutants. S15 complemented mutations in the junction loop in each of the partially functional mutants. Nonfunctional mutants were not complemented by over-expression of S15.We used single molecule Fluorescence Resonance Energy Transfer (smFRET) to study the Mg2+ and S15 induced conformational changes occurred for all types of mutants and WT loops. Comparison of the structural dynamics of these mutants and WT sequence in the presence and absence of S15 revealed specific sequence and structural motifs in the junction loop that are important in ribosome function.