E. coli RsmF activity depends on prior modification of 16S rRNA helix 44.

RsmF activity on hypomethylated 30S subunits in Escherichia coli is highly dependent on prior modification of 16S rRNA helix 44 (h44), in vitro and in vivo.

• Why it matters: The rRNA methylation cluster around the 30S decoding center likely forms during subunit biogenesis, but the required modification order for enzyme recognition was unknown.
• What they did: Using hypomethylated 30S subunits from rsmH/I/E/F-deleted Escherichia coli strains, the study tested RsmF dependence on h44 and partially rescued activity with prior in vitro methylation by RsmE and RsmH.
• The result: Structure probing (selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling) and molecular dynamics simulations show that missing m4C1402 (RsmH) and m3U1498 (RsmE) alters 16S rRNA structure/dynamics, restricting RsmF action and revealing an order of operations.