The PRC-barrel domain of the ribosome maturation protein RimM mediates binding to ribosomal protein S19 in the 30S ribosomal subunits

Abstract

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes. A DeltarimM mutant is defective in 30S maturation and accumulates 17S rRNA. To study the interaction of RimM with the 30S and its involvement in 30S maturation, RimM amino acid substitution mutants were constructed. A mutant RimM (RimM-YY-->AA), containing alanine substitutions for two adjacent tyrosines within the PRC beta-barrel domain, showed a reduced binding to 30S and an accumulation of 17S rRNA compared to wild-type RimM. The (RimM-YY-->AA) and DeltarimM mutants had significantly lower amounts of polysomes and also reduced levels of 30S relative to 50S compared to a wild-type strain. A mutation in rpsS, which encodes r-protein S19, suppressed the polysome- and 16S rRNA processing deficiencies of the RimM-YY-->AA but not that of the DeltarimM mutant. A mutation in rpsM, which encodes r-protein S13, suppressed the polysome deficiency of both rimM mutants. Suppressor mutations, found in either helices 31 or 33b of 16S rRNA, improved growth of both the RimM-YY-->AA and DeltarimM mutants. However, they suppressed the 16S rRNA processing deficiency of the RimM-YY-->AA mutant more efficiently than that of the DeltarimM mutant. Helices 31 and 33b are known to interact with S13 and S19, respectively, and S13 is known to interact with S19. A GST-RimM but not a GST-RimM(YY-->AA) protein bound strongly to S19 in 30S. Thus, RimM likely facilitates maturation of the region of the head of 30S that contains S13 and S19 as well as helices 31 and 33b.

FIGURE 1.

Amino acid sequence alignment of RimM from different eubacterial species, the malaria mosquito Anopheles gambiae, and Arabidopsis thaliana. Eco, Escherichia coli; Bsu, Bacillus subtilis; Vpa, Vibrio parahaemolyticus; Nme, Neisseria meningitidis; Ath, Arabidopsis thaliana; Syn, Synechocystis sp. Strain PCC6803; Cte, Clostridium tetani; Aae, Aquifex aeolicus; Cht, Chlorobium tepidum; Fnu, Fusobacterium nucleatum; Mlo, Mesorhizobium loti; Mle, Mycobacterium leprae; Dra, Deinococcus radiodurans; Aga, Anopheles gambiae; Bbu, Borrelia burgdorferi; Hpy, Helicobacter pylori. The numbering to the left of the top row indicates the position in the respective sequence of the first amino acid shown. Amino acid substitutions were isolated for the positions indicated by arrows. The * indicates the position of the N84K substitution suppressing RimM-YY→AA.

FIGURE 2.

The effect of S13Δ89-99 and S19H83Y on the growth rate of the ΔrimM and RimM-YY→AA mutants grown in LB medium at 37°C. The growth rate was determined in four independent experiments and is expressed as k (= ln2/g, where g is the mass doubling time in h). The variation between the experiments is shown as error bars.

FIGURE 3.

The effect of S13Δ89-99 and S19H83Y on polysome profiles of the ΔrimM and RimM-YY→AA mutants grown in LB medium at 37°C. Different ribosome particles are marked above corresponding peaks in A.

FIGURE 4.

The effect of suppressor mutations in helices 31 and 33b of 16S rRNA on the growth rate of the RimM-YY→AA (A) and ΔrimM (B) mutants in LB medium at 37°C. The growth rate was determined in three independent experiments and is expressed as k (= ln2/g, where g is the mass doubling time in h). The variation between the experiments is shown as error bars.

FIGURE 5.

Mutations in helices 31 and 33b of 16S rRNA that suppress the slow growth of the RimM-YY→AA and ΔrimM mutants JML068 and JML114.

FIGURE 6.

The effect of different suppressor mutations on the 16S rRNA processing deficiency of rimM mutants. The 5′-end of 16S rRNA was determined by primer extension analysis. RIII indicates a primer extension product of 179 nt corresponding to 17S rRNA processed at the RNase III site 115 nt upstream of the 5′-end of mature 16S rRNA. M indicates a product of 64 nt corresponding to the 5′-end of mature 16S rRNA. The sequencing ladder shown is of rrnC in plasmid pHK-rrnC⁺ and was obtained by using the same primer as for the primer extension reactions. (A) The effect of S13Δ89-99 and S19H83Y on the 16S rRNA processing in ΔrimM and RimM-YY→AA mutants. (B) The effect of 16S rRNA mutations on 16S rRNA processing in ΔrimM and RimM-YY→AA mutants.

FIGURE 7.

Analysis of cellular components copurifying with GST-RimM hybrid proteins at 4°C. (A) A cell extract of strain GOB191/ pJML005 (ΔrimM/GST-RimM) was prepared by freeze-thawing, and the GST-RimM hybrid protein in the extract was adsorbed to Glutathione Sepharose 4B, washed at the indicated molar concentrations of NaCl and finally eluted by reduced glutathione, all at 4°C. FT, flow-through; E, eluate. (B) As in A, but the strain used was GOB191/ pJML051 [ΔrimM/GST-RimM(YY→AA)]. (C) Primer extension analysis of 16S rRNA prepared from the flow-through and eluate from A, and from 30S subunits obtained after sucrose gradient centrifugation of a total cell extract of the wild-type strain MW100.

FIGURE 8.

Analysis of cellular components copurifying with GST-RimM hybrid proteins at 21°C. Cellular extracts were prepared by sonication and the GST-RimM hybrid proteins were purified as described in the Figure 7 ▶ legend. Similar results were obtained when extracts were prepared by freeze-thawing. (A) Strain GOB191/pJML005 (ΔrimM S19-wt/GST-RimM). The proteins indicated in the lane with the 0.6 M NaCl wash fraction were identified as 30S subunit r-proteins by MALDI mass spectrometry: 1, S2; 2, S3; 3, S4; 4, S7; 5, S5; 6, S9; 7, S11 and S13; 8, S10; and 9, S17. In addition, one weak band above that containing S2 was identified as r-protein L2. (B) Strain GOB191/pJML051 [ΔrimM S19-wt/GST-RimM(YY→AA)]. (C) Strain MW158/pJML005 (ΔrimM S19H83Y/GST-RimM). (D) Strain MW158/pJML051 [ΔrimM S19H83Y/GST-RimM(YY→AA)].

FIGURE 9.

Alterations in the E. coli 30S subunit (indicated in the model for the 30S subunit of Thermus thermophilus) that suppress rimM mutations. The structure of the 30S subunit is from Wimberly et al. (2000) and was retrieved from the Protein Data Bank (PDB no. 1FJF). Only part of the structure is shown. Helices 31 and 33b of 16S rRNA are presented as stick models in green except for the positions with suppressor mutations, which are in space-fill models. R-protein S13 is shown in a blue ribbon model except for a region in gray, which contains the alterations that suppress rimM mutations. S19 is shown in a yellow ribbon model with the most carboxy terminal amino acid, R81, of the determined structure, colored red. Thus, the alteration, H83Y, in S19 that suppressed RimM-YY→AA is in a position just outside of the determined structure.