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

Bacterial ribosomal RNA (rRNA) methylations are important for accurate translation. Four distinct methylations incorporated by RsmE, RsmF, and RsmH/ RsmI form a cluster of three modified 16S rRNA nucleotides (m3U1498, m5C1407, and m4Cm1402) surrounding the decoding center of the 30S subunit. Given their common substrate requirement of a late-stage intermediate 30S subunit, these enzymes likely act contemporaneously during subunit biogenesis, but whether there exists a required modification order is unknown. Here, using hypomethylated 30S subunits obtained from a collection of rsmH/I/E/F-deleted Escherichia coli strains, we identify RsmF activity to be highly dependent on prior modification of h44 both in vitro and in E. coli. RsmF activity on hypomethylated 30S subunits could be partially rescued by prior in vitro methylation using RsmE and RsmH, indicating that incorporation of these methyl groups directly shapes h44 for recognition by RsmF. RNA structure probing using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling and molecular dynamics simulations reveal specific alterations in 16S rRNA structure and dynamics in the absence of the m4C1402 (RsmH) and m3U1498 (RsmE) modifications that likely restrict RsmF action. These studies thus uncover a previously unappreciated "order of operations" for 16S rRNA modification during ribosome biogenesis with important implications for studies on the collective functions of these modifications.