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RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

Figure 2

Mapping the RsfA binding site on ribosomal protein L14.

(A) L14 in the context of the 3D structure of the 50S ribosomal subunit (a) (PDB: 2AWB) [14]. (b) Conserved residues of L14: magenta (highly conserved), grey (moderately conserved), turquoise (little or no conservation). (c) Mutated residues for interaction epitope mapping (red or green); residues involved in (red colors) and not involved (green colors) in RsfA-binding based on results from subfigure (C). (d) Residues of L14 highlighted that are involved in formation of intersubunit bridges with the 16S rRNA of the 30S subunit (bridge B5 (green colors), bridge B8 (red colors)) [15]. (B) A docking model of L14 on the E. coli 50S subunit with bound RsfA. Critical L14 residues that mediate RsfA interaction (or that contact 16S rRNA) are colored in red according to A(c) and A(d). When RsfA is bound to L14 on a 50S subunit, 30S subunit joining is sterically blocked, clearly visible in B(b) as shown by the structural overlap of RsfA (dark blue) and the 30S subunit. A model of the ribosome with bound RsfA is available as Dataset S1. (C) L14 interaction epitope mapping. Amino acids (see Figure 2A(c)) were mutated to alanine and the constructs tested by Y2H experiments. WT, wild type L14 construct; mutated residues and their positions are indicated. In the experiment, all bait constructs were simultaneously tested for reporter gene self-activation. No construct resulted in self-activation (data not shown). T97A, R98A, or K114A mutations (highlighted by arrows) abolished or weakened RsfA binding as indicated by 3-AT titrations; all other tested L14 mutation constructs are comparable to wild type L14.

Figure 2

doi: https://doi.org/10.1371/journal.pgen.1002815.g002