Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation

Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2'-O-methylation of U2552 in 235 rRNA by methyl-transferase RrmJ is a crucial step in late-stage assembly of the 505 subunit. Its absence results in severe growth defect and marked accumulation of pre5OS assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre5OS particles isolated from an Escherichia coli Delta rrmJ strain. These assembly intermediates (solved at 3.2 to 3.8 angstrom resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 505 subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 505 subunit from Delta rrmJ strain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the Delta rrmJ 50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the Delta rrmJ 70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.