Dissecting the in vivo assembly of the 30S ribosomal subunit reveals the role of RimM and general features of the assembly process

Abstract

Ribosome biogenesis is a tightly regulated, multi-stepped process. The assembly of ribosomal subunits is a central step of the complex biogenesis process, involving nearly 30 protein factors in vivo in bacteria. Although the assembly process has been extensively studied in vitro for over 40 years, very limited information is known for the in vivo process and specific roles of assembly factors. Such an example is ribosome maturation factor M (RimM), a factor involved in the late-stage assembly of the 30S subunit. Here, we combined quantitative mass spectrometry and cryo-electron microscopy to characterize the in vivo 30S assembly intermediates isolated from mutant Escherichia coli strains with genes for assembly factors deleted. Our compositional and structural data show that the assembly of the 3'-domain of the 30S subunit is severely delayed in these intermediates, featured with highly underrepresented 3'-domain proteins and large conformational difference compared with the mature 30S subunit. Further analysis indicates that RimM functions not only to promote the assembly of a few 3'-domain proteins but also to stabilize the rRNA tertiary structure. More importantly, this study reveals intriguing similarities and dissimilarities between the in vitro and the in vivo assembly pathways, suggesting that they are in general similar but with subtle differences.

Figure 1.

Phenotypes of the ΔrimM and ΔrbfAΔrsgA strains. (A) Spot assay showing that both the ΔrimM (Δ) and ΔrbfAΔrsgA (ΔΔ) strains grow slowly, compared with the wild-type strain (WT). (B) Ribosome profile analysis of the A19, ΔrimM and ΔrbfAΔrsgA strains. The profile curves of the WT, Δ and ΔΔ strains are colored in black, green and red, respectively. Deletion of RimM or a combination of RbfA and RsgA causes an accumulation of immature 30S subunits. Both experiments indicate that deletion of RimM is more deleterious.

Figure 2.

Compositional characterization of the immature 30S subunits. Composition of mature and immature 30S subunits from the A19 ΔrimM (Δ) and ΔrbfAΔrsgA (ΔΔ) strains was analyzed in both the RNA and protein levels. (A) RNA gel analysis of the rRNAs in the Δ and ΔΔ samples. (B) Tricine-SDS–PAGE analysis of the protein composition of the immature 30S subunits. (C) QMS analysis of the protein composition in the Δ and the ΔΔ samples. Error bars show standard deviations. The difference in protein ratio between the Δ and ΔΔ samples was subjected to a one-tailed t-test, which reports a significant difference for S10, S14, S13, S3, S12, S5, S4, S7 (P < 0.01), S19 and S6 (P < 0.05). (D) The relative protein ratios of the ΔΔ sample to the Δ sample (ΔΔ/Δ) are plotted against the ratios of the Δ sample to the mature one (Δ/mature). (E) Atomic structure of the mature 30S subunit (38) viewed from the inter-subunit and solvent sides, with proteins in the top-left part of (D) colored in green.

Figure 3.

Overview of the five cryo-EM structures of the immature 30S subunits from the ΔrimM strain. The five density maps (A–E or F–J, respectively) are displayed in transparent surface representation, superimposed with flexibly fitted crystal structures in cartoon representation. For each map, both the inter-subunit view (A–E) and the solvent view (F–J) are displayed. The 16S rRNA, S2, S3 and the rest 30S subunit proteins are painted in blue, green, red and purple, respectively. Deviations of the 16S rRNA backbones in the fitted model from that of the mature 30S subunit are colored as indicated by the scale to form the temperature maps (K–O).

Figure 4.

RimM preferentially binds to the immature 30S subunits from the ΔrimM strain. Immature 30S subunits from the ΔrimM strain and mature 30S subunits from the 70S ribosomes were incubated with or without a 30-fold excess of RimM. The mixtures were pelleted by centrifugation. The pellets (P) and the supernatants (S) were separated and resolved by SDS–PAGE. RimM alone was centrifuged as a control. The asterisk denotes the weak binding of RimM to the immature 30S subunits.

Figure 5.

Overview of the five cryo-EM structures of the immature 30S subunits treated with RimM. The five density maps (A–E or F–J, respectively) are displayed in transparent surface representation, superimposed with flexibly fitted crystal structures in cartoon representation. For each map, both the inter-subunit view (A–E) and the solvent view (F–J) are displayed. The 16S rRNA, S2, S3 and the rest 30S subunit proteins are painted in blue, green, red and purple, respectively. Deviations of the 16S rRNA backbones in the fitted model from that of the mature 30S subunit are colored as indicated by the scale to form the temperature maps (K–O).

Figure 6.

Mechanistic model of the RimM function in the in vivo assembly of the 3′-domain. (A) The head domain of the 30S subunit viewed from the inter-subunit side. (B) Same as (A), with a 70° rotation around Y-axis. The h33b, h31 and the rest of the 16S rRNA are painted in cyan, wheat and blue, respectively. The C-terminal domain of RimM (CTD), N-terminal domain of RimM (NTD), S7, S13, S19 and S14 are painted in magenta, orange, red, yellow, green and purple, respectively. (C) RimM, RbfA and RsgA act at different checkpoints during the in vivo assembly. The deficiency of assembly factors diverts the assembly into less efficient branches (colored dash lines) and causes accumulation of a set of closely related intermediates (colored boxes). The ribosomal protein levels in the three sets of in vivo intermediates are displayed in the gray scale. The data of the intermediates from a ΔrsgA strain is from a previous study (50). The large conformational differences among the three sets of in vivo intermediate were also shown in cartoon: the ΔrimM one (red) with a dramatically rotated head domain and a disordered helix 44; the ΔrbfAΔrsgA one (green) with a disordered helix 44 only; the ΔrsgA one (blue) with a well-resolved helix 44.