Poster abstracts

Poster number 34 submitted by Rajan Lamichhane

Single Molecule FRET to Explore Protein-RNA Interactions in the Central Domain of the 30S Ribosomal Subunit

Rajan Lamichhane (Department of Chemistry, Wayne State University, Detroit, MI 48202), Kris A. Baker (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), Philip R. Cunningham (Department of Biological Sciences, Wayne State University, Detroit, MI 48202), David Rueda (Department of Chemistry, Wayne State University, Detroit, MI 48202)

Abstract:
The central domain of the 16S rRNA contains several protein binding sites. S15 is a central domain primary binding protein that has been shown to trigger a conformational change in the rRNA.1 This conformational change allows other central domain binding proteins to bind to the central domain causing a cascade of changes resulting in the functional structure of the central domain. Previous biochemical and structural studies have revealed two regions that are minimally needed for binding S15 in vitro. One of the regions is the junction of helices 20, 21, and 22 in 16S rRNA, which includes nucleotides 652-654 and 752-754 plus two or three base pairs in each helix to maintain the junction structure.

To identify functionally important sequence and structural elements within the junction loop, nucleotides 652-654 and 752-754 were subjected to saturation mutagenesis and functional mutants were selected and analyzed. To determine if S15 binding was affected by mutations in the junction loop, S15 was cloned and over-expressed with the junction mutations. S15 complemented mutations in the junction loop in each of the partially functional mutants. Nonfunctional mutants were not complemented by over-expression of S15.

Individual mutants were analyzed using single molecule Fluorescence Resonance Energy Transfer (smFRET) in vitro. Comparison of the structural dynamics of these mutants to that of the WT sequence in the presence and absence of S15 has revealed specific sequence and structural motifs in the junction loop that are important in ribosome function.

References:
1. Agalarov, S. C., Sridhar Prasad, G., Funke, P. M., Stout, C. D. & Williamson, J. R. (2000). Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain. Science 288, 107-13.

Keywords: S15, Ribosome, smFRET