AptERA 2 targets ERA from Staphylococcus aureus and limits GTP hydrolysis

Ribosome assembly is a multistep process that ensures a functional ribosome structure. The molecular mechanism that ribosome­associated GTPases (RA­GTPases) use to enhance ribosome assembly accuracy, remains largely to be elucidated. Here, we use systematic evolution of ligands by exponential enrichment (SELEX), followed by sequencing, comprehensive bioinformatics analysis, and biochemical characterization to identify aptamers that target the RA-GTPase ERA of Staphylococcus aureus . ELONA and thermophoresis assays show that the Apt _ERA 2 interaction with ERA is in the 200 nM range of affinity, displays a high level of specificity, and depends on the target structure. Docking to ERA suggests that Apt _ERA 2 interacts with the protein's KH domain, consistent with the aptamer's similarities with helix 45 of the 16S rRNA. Apt _ERA 2 did not interact with a similar RA-GTPase RbgA, conserved at the GTPase core but lacking the KH domain, confirming that the aptamer recognizes and binds the KH domain of ERA. This interaction leads to a significant reduction of 30S-dependent GTP hydrolysis, indicative of allosteric modulation of the enzyme activity or limiting the KH domain interaction with the 3’ end of the 16S rRNA rather than directly blocking GTP binding. Altogether, this work highlights the versatility of aptamers as tools to understand the complex processes of ribosome biogenesis further, offering new insights into bacterial protein synthesis mechanisms.