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

Abstract

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.

FIGURE 1.

A comparison of pseudouridine modification activity of wild-type RluD (WT) and ΔS4 RluD (ΔS4) on ΔBED 50S subunits with the activity of WT RluD on full-length 23S RNA and domain IV of 23S RNA, as monitored by tritium release and Ψ sequencing analyses. (A) Time course of pseudouridylation activity on 50S subunits extracted from a ΔBED strain and synthetic full-length 23S RNA and domain IV of 23S RNA. Reactions were carried out as described in Materials and Methods. Reaction mixtures contained 200 nM substrate RNA and the indicated amounts of purified His-tagged protein. The amount of pseudouridine modification was monitored by the tritium release assay. (•) 5 nM WT RluD on 50S, (△) 2 μM ΔS4 RluD on 50S, (□) 20 nM WT RluD on full-length 23S RNA, (⨯) 20 nM WT RluD on domain IV of 23S RNA. The average of three experiments is shown. Error bars represent standard deviation. (B) Ψ sequencing analysis of RNA extracted from 50S subunits from A at the earliest (0′) and last (60′) time points. RNA was reacted with (+) or without (−) CMC following the standard sequencing protocol. While several stops can be seen, only CMC-dependent changes in intensity are indicative of Ψ. The three RluD target uridines (1911, 1915, and 1917) are marked by black arrowheads.

FIGURE 2.

Pseudouridine modification activity of wild-type RluD, ΔS4 RluD, and wild-type RluE on synthetic 23S RNA, as monitored by tritium release and/or Ψ sequencing analyses. (A) Time course of pseudouridylation activity on synthetic RNA at different enzyme concentrations. Reactions were carried out as described in Materials and Methods. Reaction mixtures contained 200 nM RNA and the indicated amounts of purified His-tagged protein. The extent of pseudouridine modification was monitored using the tritium release assay. (——) WT RluD, (– – –) ΔS4 RluD. (•) 20 nM WT RluD, (▪) 200 nM WT RluD, (▴) 2 μM WT RluD. (○) 20 nM ΔS4 RluD, (□) 200 nM ΔS4 RluD, (△) 2 μM ΔS4 RluD. The average of three experiments is shown. Error bars represent standard deviation. (B) Ψ sequencing analysis of RNA extracted from reactions in A at lowest (20 nM) and highest (2 μM) concentrations of wild-type RluD (WT) and ΔS4 RluD (ΔS4) at the earliest (0′) and last (60′) time points. RNA was reacted with (+) or without (−) CMC following the standard sequencing protocol. The three RluD target uridines (1911, 1915, and 1917) are marked by black arrowheads. No change in intensity is evident between (+) and (−) CMC lanes for ΔS4, signifying the lack of any modification at the three sites. (C) Ψ sequencing analysis of synthetic 23S RNA extracted from pseudouridylation reactions at lowest (20 nM) and highest (2 μM) concentrations of wild-type RluE and ΔS4 RluD at the earliest (0′) and last (60′) time points. RNA was reacted with (+) or without (−) CMC following the standard sequencing protocol. The RluE target uridine (2457) is marked by a black arrowhead.

FIGURE 3.

RluD is specific to the 50S subunit. The pseudouridylation activity of increasing amounts (20 nM [open], 200 nM [gray], and 2 μM [stippled]) of purified, His-tagged, full-length RluD was compared on different RNA species extracted from ΔBED mutant cells. The substrates were maintained at a constant concentration of 200 nM of RNA. The substrates included 16S RNA (16S), 30S subunits (30S), 23S RNA (23S), 50S subunits (50S), and 70S ribosomes (70S). The 16S and 23S RNA substrates were extracted from their respective subunits as described in Materials and Methods. The reactions were carried out for an hour as described in Materials and Methods. An average of two experiments is shown. Error bars represent standard deviation.

FIGURE 4.

Model of RluD docked on the H69 stem–loop in the 50S subunit, poised to modify U1915. A stereo view shown focuses on H69 in the 50S (gray cartoon) with RluD represented by a colored ribbon. Full-length RluD (PDB ID: 2IST) was manually docked on to the H69 stem–loop of the E. coli 50S subunit (PDB ID: 2AWB) as described in Materials and Methods. The catalytic Asp (D139) of RluD and U1915 of 23S RNA are depicted as red and black stick models, respectively. Three residues (73–75) connecting the S4-like domain to the catalytic domain of RluD are not shown, since they are missing in the 2IST structure.