Evidence that ribosomal protein bS21 is a component of the OLE ribonucleoprotein complex

OLE RNAs represent a large and highly structured noncoding RNA (ncRNA) class that is mostly found in Gram-positive extremophiles and/or anaerobes of the Bacillota phylum. These ~600-nucleotide RNAs are among the most structurally complex and well-conserved large ncRNAs whose precise biochemical functions remain to be established. In Halalkalibacterium halodurans, OLE RNA is involved in the adaptation to various unfavourable growth conditions, including exposure to cold (≤20°C), ethanol (≥3% [v/v]), excess Mg²⁺ (≥4 mM), and non-glucose carbon/energy sources. OLE forms a ribonucleoprotein (RNP) complex with the OLE-associated proteins A, B and C, which are known to be essential for OLE RNP complex function in this species. Bacteria lacking OLE RNA (Δole) or a functional OLE RNP complex exhibit growth defects under the stresses listed above. Here, we demonstrate that ribosomal protein bS21 is a natural component of the OLE RNP complex and we map its precise RNA binding site. The presence of bS21 results in a conformational change in OLE RNA resembling a k-turn substructure previously reported to be relevant to the function of the OLE RNP complex. Mutational disruption of the bS21 protein or its OLE RNA binding site results in growth inhibition under cold and ethanol stress to the same extent as the deletion of the gene for OLE RNA. These findings are consistent with the hypothesis that bS21 is a biologically relevant component of the OLE RNP complex under a subset of stresses managed by the OLE RNP complex.