In this work, we show that 23S rRNA lacking the SRL can be efficiently reconstituted into 50S subunits, which exhibit full peptidyl transferase (PT) activity. Using cryo-EM and proteomic methods, we also further characterize affinity-purified SRL particles isolated from cells. Collectively, these data suggest that the SRL is needed to promote the dissociation of several AFs during 50S subunit biogenesis in the cell.