Talk abstracts

Talk on Saturday 09:00-09:20am submitted by Kris Baker

Protein-RNA Interactions in the Central Domain of 30 S ribosomal subunit

Kris A. Baker (Department of Biological Sciences, Wayne State University), James R. Williamson (Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute), Philip R. Cunningham (Department of Biological Sciences, Wayne State University)

Abstract:
Assembly of functional 30 S subunits is dependent on ribosomal protein (rprotein) - ribosomal RNA (rRNA) interactions that facilitate and stabilize folding of 16 S rRNA into the active conformation. Binding sites for several rproteins are located in the central domain of 16 S rRNA. S15, a central domain primary binding protein, has been shown to trigger a conformational change in the rRNA. This conformational change allows other central domain proteins - S6, S18, S11 and, S21 - to bind to the region causing a cascade of changes resulting in the functional structure of the central domain.

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. The junction includes nucleotides 652-654 and 752-754 plus two or three base pairs in each helix for maintaining the junction structure. All junction nucleotides except 653 are highly conserved in bacteria implying these nucleotides are functionally important. Mutagenesis studies suggest that the junction plays a role in S15 recognition of 16 S rRNA. In particular, 752 mutants have decreased affinity for S15.

The interaction of the junction loop (652-654 and 752-754) with S15 does not appear to be base-specific; instead, S15 binds to the backbone of nucleotides 752-754. Interactions between nucleotides 652-654 and 752-754 create a structure that places the backbone in the proper position to bind S15. Conservation of junction nucleotides possibly occurs because single nucleotide changes would disrupt S15 binding. To identify alternative functional junction loop sequences and the common structural motif, a saturation mutagenesis experiment of nucleotides 652-654 and 752-754 was performed.

Nucleotides 652-654 and 752-754 were randomly mutated and functional sequences were selected. Sixty-one unique mutants were isolated in the selection, sequenced, and assayed for function. No nucleotide was excluded at any position. The wild-type nucleotide appears to be preferred at each position except at 653. 653 had a non-random distribution of nucleotides with adenosine being preferred. Single-site mutagenesis at 653 showed no preference for nucleotide identity.

Keywords: ribosomal protein S15, 30 S subunit central domain