RluD, a highly conserved pseudouridine synthase, modifies 50S subunits more specifically and efficiently than free 23S rRNA

Pseudouridine modifications in helix 69 (H69) of 23S ribosomal RNA are highly conserved among all organisms. H69 associates with helix 44 of 16S rRNA to form bridge B2a, which plays a vital role in bridging the two ribosomal subunits and stabilizing the ribosome. The three pseudouridines in H69 were shown earlier to play an important role in 50S subunit assembly and in its association with the 30S subunit. In Escherichia coli, these three modifications are made by the pseudouridine synthase, RluD. Previous work showed that RluD is required for normal ribosomal assembly and function, and that it is the only pseudouridine synthase required for normal growth in E. coli. Here, we show that RluD is far more efficient in modifying H69 in structured 50S subunits, compared to free or synthetic 23S rRNA. Based on this observation, we suggest that pseudouridine modifications in H69 are made late in the assembly of 23S rRNA into mature 50S subunits. This is the first reported observation of a pseudouridine synthase being able to modify a highly structured ribonucleoprotein particle, and it may be an important late step in the maturation of 50S ribosomal subunits.