Ribosome‐associated factor Y adopts a fold resembling a double‐stranded RNA binding domain scaffold

Abstract

Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl-tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112-residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a babbba topology and represents a compact twolayered sandwich of two nearly parallel a helices packed against the same side of a four-stranded b sheet. The 23 C-terminal residues of the protein are disordered. Longrange angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C-terminal region of helix 1 and the loop linking this helix with strand b2 show significant conformational exchange in the ms-ls time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C-terminal segments of both a helices, which most probably constitutes an RNA binding site. The observed babbba topology of pY resembles the abbba topology of double-stranded RNA-binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA-binding site in pY does not coincide with the RNA-binding surface of the dsRBDs.Keywords: backbone dynamics; dsRBD; molecular alignment; residual dipolar couplings; RNA-binding domain.Protein expression is finely tuned in the cell allowing for adaptations to various environmental changes. The regulation strategies adopted by the cell encompass almost every aspect of the protein production process, including mRNA transcription, translation in the ribosome and protein degradation. Ribosome activity constitutes an important target in the control of protein expression. Protein Y, the product of yfiA gene, is a ribosome-associated protein present in E. coli and many other bacteria [1][2][3]. pY was earlier assigned to the r 54 modulation protein family based on the observation that mutations in the related downstream r 54 gene cause an increase in the level of expression from r 54 -dependent promoters [4].pY (also called ribosome associated inhibitor, RaiA) [2] was detected in the ribosome fraction during environmental stress, as a consequence of either low temperature [2] or excessive cell density [2,3]. Furthermore, pY binds to the small ribosomal subunit in an Mg 2+ -dependent manner and becomes less exposed to solvent upon association of the small and large ribosomal subunits. This suggests an intersubunit position for pY in the 70S ribosome and may explain its ribosome stabilization effect [1]. pY inhibits translation most probably by interfering with the binding...

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