Lamotrigine-mediated rescue of RsgA-deficient Escherichia coli reveals another role of IF2 in ribosome biogenesis

Abstract

RsgA (small ribosomal subunit, 30S, GTPase), a late-stage biogenesis factor, releases RbfA from 30S-RbfA complex. Escherichia coli ΔrsgA (deleted for rsgA) shows a slow growth phenotype and an increased accumulation of 17S rRNA (precursor of 16S rRNA) and the ribosomal subunits. Here, we show that the rescue of the ΔrsgA strain by multicopy infB (IF2) is enhanced by simultaneous overexpression of initiator tRNA (i-tRNA), suggesting a role of initiation complex formation in growth rescue. The synergistic effect of IF2/i-tRNA is accompanied by increased processing of 17S rRNA (to 16S), and protection of the 16S rRNA 3'-minor domain. Importantly, we show that an IF2-binding anticonvulsant drug, lamotrigine (Ltg), also rescues the ΔrsgA strain growth. The rescue is accompanied by increased processing of 17S rRNA, protection of the 3'-minor domain of 16S rRNA, and increased 70S ribosomes in polysome profiles. However, Ltg becomes inhibitory to the ΔrsgA strain whose growth was already rescued by an L83R mutation in rbfA. Interestingly, like wild-type infB, overproduction of Ltg^RinfB alleles (having indel mutations in their domain II) also rescues the ΔrsgA strain (independent of Ltg). Our observations suggest the dual role of IF2 in rescuing the ΔrsgA strain. First, together with i-tRNA, IF2 facilitates the final steps of processing of 17S rRNA. Second, a conformer of IF2 functionally compensates for RsgA, albeit poorly, during 30S biogenesis.

Importance: RsgA is a late-stage ribosome biogenesis factor. Earlier, infB (IF2) was isolated as a multicopy suppressor of the Escherichia coli ΔrsgA strain. How IF2 rescued the strain growth remained unclear. This study reveals that (i) the multicopy infB-mediated growth rescue of E. coli ΔrsgA and the processing of 17S precursor to 16S rRNA in the strain are enhanced upon simultaneous overexpression of initiator tRNA and (ii) a conformer of IF2, whose occurrence increases when IF2 is overproduced or when E. coli ΔrsgA is treated with Ltg (an anticonvulsant drug that binds to domain II of IF2), compensates for the function of RsgA. Thus, this study reveals yet another role of IF2 in ribosome biogenesis.

Keywords: 3GC base pairs; RbfA; initiation factor 2; initiator tRNA; rRNA maturation; ribosome.

Fig 1

(A) Dilution spotting plate assay showing growth of serially diluted (10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) log phase cultures of KL16 and KL16ΔrsgA strains harboring empty plasmid pACDH (Tet^R) and pEmpty (ColE1, Amp^R) and plasmid borne copies of IF2, i-tRNA alone, or together (IF2 and i-tRNA) at 28°C for 36 h. pACDH having ACYC origin of replication expressing IF2 and pEmpty having ColE1 origin of replication expressing metY (i-tRNA) are compatible with each other. (B) Northern blot showing 17S precursor and 16S rRNA in E. coli KL16 and its ΔrsgA derivative with overexpressed IF2 and i-tRNA alone or together (IF2 and i-tRNA) at 28°C and quantification of relative change of 17S rRNA in different background in reference to KL16 having empty vector. Band intensities were quantified using Multi Gauze software V 2.7 (Fujifilm). Ratios (17S/16S) were calculated by dividing the band intensity of 17S precursor with that of the 16S rRNA individually for each of the samples. The values of the relative ratios (used for plotting) were obtained by dividing each of the 17S/16S ratios with the value obtained for the KL16 having empty vector. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed using one way analysis of variance (ANOVA) (GraphPad Prism, version 8.0.2). Statistical significance, *P < 0.05 and ***P < 0.001.

Fig 2

(A) Dilution spotting plate assay showing growth of serially diluted (10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) log phase cultures of ΔrsgA strains harboring empty plasmid pACDH (Tet^R) and pEmpty (Amp^R) plasmid borne genes of IF2 and i-tRNA alone or together (IF2 and i-tRNA) at 28°C for 36 h in the presence of increasing amounts of Ltg. (B) Growth curve showing effect of increasing concentrations of Ltg (20, 40, 80, and 160 µM) on ΔrsgA mutant strains at 28°C. The line joins the mean of three independent replicates, and error bars represent the standard deviation. (C) Northern blots showing level of 17S precursor and 16S rRNA in the presence of increasing Ltg (20, 40, 80, and 160 µM) on ΔrsgA mutant strains with genomic copy of IF2_WT. The values of the relative ratios (used for plotting) were calculated as described in Fig. 1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed using one way ANOVA (GraphPad Prism, version 8.0.2). Statistical significance, *P < 0.05 and ***P < 0.001.

Fig 3

(A) Schematic showing deletion (IF2_Δ1, IF2_Δ8, and IF2_Δ17) and duplication (IF2-8×2) of amino acids present at N terminal region in subdomain II of IF2. (B) Northern blot showing levels of 17S precursor (using a probe complementary to the 3′-end sequences of the 17S rRNA) and 16S rRNA (using a probe against the internal region of 16S rRNA) in the isogenic derivatives of ΔrsgA strain with genomic copies of IF2_WT, IF2_Δ1, and IF2_Δ8 genes under no Ltg (control) or Ltg (treated) conditions (i) and their quantification (ii). The values of the relative ratios (used for plotting) were calculated as described in Fig. 1. Data are shown as mean ± SD of three independent experiments. Statistical analysis was performed using one way ANOVA (GraphPad Prism, version 8.0.2). Statistical significance, *P < 0.05 and ***P < 0.001. (C) Agarose gel electrophoresis showing 17S, 16S, and 16S* rRNAs in isogenic derivatives of the ΔrsgA strain with genomic copy of IF2_WT, IF2_Δ1, and IF2_Δ8 strains under no Ltg or Ltg- (40 µM) treated conditions. (D) Polysome profiles showing levels of 30S, 50S, and 70S of ΔrsgA strain under untreated (i), and Ltg- (40 µM) treated conditions (ii).

Fig 4

(A) Dilution spotting plate assay showing growth of serially diluted (10⁻¹, 10⁻², 10⁻³, and 10⁻⁴) log phase cultures of ΔrsgA strains harboring empty plasmid pACDH (Tet^R) and plasmid-borne genes of IF2_WT, IF2_Δ1, IF2_Δ8, IF2_Δ17, and IF2-8×2 at 28°C (I) and SDS-PAGE showing expression of IF2_WT, IF2_Δ1, IF2_Δ8, IF2_Δ17, and IF2-8×2 at mid log phase, grown at 28°C in ΔrsgA strain (ii). (B and C) Agarose gel electrophoresis showing 17S, 16S, and 16S* rRNAs of ΔrsgA strains harboring empty plasmid pACDH (Tet^R) and plasmid-borne genes of IF2_WT, IF2_Δ1, IF2_Δ8, IF2_Δ17, and IF2-8×2 and ΔrsgA strains harboring empty plasmid pACDH (Tet^R) and pEmpty (Amp^R) plasmid-borne genes of IF2, i-tRNA alone, or together (IF2 and i-tRNA) at 28°C, respectively.

Fig 5

(A) Growth analysis of KL16 ΔrsgA strain harboring genomic copies of RbfA or RbfA (L83R) with or without Ltg (40 µM) at 28°C. (B) Plate dilution spotting assay showing Ltg effect on KL16 ΔrsgA strains with genomic copies of RbfA or RbfA (L83R) genes.

Fig 6

(A) The 30S pre-initiation complex (30S PIC) formation with 30S (having 17S precursor), mRNA, IFs (IF1, IF2, and IF3), and formyl-aminoacyl-i-tRNA, which rearranges into 30S IC, joins 50S to form 70S PIC, which then leads 70S complex (elongation competent) upon release of IFs. Processing of the 17S precursor occurs by various RNases, as indicated following 50S joining to 30S IC. Release of any residual RbfA that may remain with some 30S molecules during this process (26, 27) has not been shown. (B) Release of RbfA from 30S by RsgA during 30S biogenesis in E. coli wild type for RsgA (left panel). Proposed roles of IF2 conformers (IF2*) facilitated either by Ltg binding (central panel) or occurring spontaneously upon overexpression of IF2 (right panel) in RbfA release in ΔrsgA strain.