A Two-Step Purification Strategy for RNA and RNA-Protein Complexes for Crystallization

In Legionella pneumophila, RocC, an RNA chaperone, and RocR, its targeted sRNA, play a critical role in repressing the development of competence at the posttranscriptional level. Understanding RocR recognition by RocC has been challenging due to the flexible nature of RNA and the difficulty of preparing it in large quantities using conventional methods. To address these issues, we engineered RNA with reduced flexibility, guided by computational predictions and validated through electrophoretic mobility shift assays (EMSAs). We established a workflow for producing large amounts of homogeneous engineered RNA, incorporating tandem anion-exchange and size-exclusion chromatography via fast protein liquid chromatography (FPLC) to achieve high-purity RNA and RNA-protein complexes. This streamlined RNA purification framework enabled successful crystallization of the sRNA RocR in complex with the RNA chaperone RocC.