An evolutionarily conserved element in initiator tRNAs prompts ultimate steps in ribosome maturation

Significance

Ribosomes are complex molecular machines requiring an intricate pathway for their biogenesis. Deficiencies in ribosome biogenesis result in inefficient and inaccurate translation, causing cellular toxicities and ribosomopathies. As ribosomes have multiple functions during the translation process, how cells ensure fidelity of the newly synthesized ribosomes for their functions has remained unclear. The immature ribosomes possess rRNAs whose ends have not been fully processed. What licenses final trimming of immature rRNAs is also unclear. Here, using Escherichia coli, we show that participation of initiator tRNA via its universally conserved three consecutive GC base pairs, in the first round of the initiation complex formation licenses the final steps of ribosome biogenesis by signaling RNases to trim the immature 16S rRNAs.

Abstract

Ribosome biogenesis, a complex multistep process, results in correct folding of rRNAs, incorporation of >50 ribosomal proteins, and their maturation. Deficiencies in ribosome biogenesis may result in varied faults in translation of mRNAs causing cellular toxicities and ribosomopathies in higher organisms. How cells ensure quality control in ribosome biogenesis for the fidelity of its complex function remains unclear. Using Escherichia coli, we show that initiator tRNA (i-tRNA), specifically the evolutionarily conserved three consecutive GC base pairs in its anticodon stem, play a crucial role in ribosome maturation. Deficiencies in cellular contents of i-tRNA confer cold sensitivity and result in accumulation of ribosomes with immature 3′ and 5′ ends of the 16S rRNA. Overexpression of i-tRNA in various strains rescues biogenesis defects. Participation of i-tRNA in the first round of initiation complex formation licenses the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA.

Ribosomes are large and the most complex of the molecular machines in cells, using up to 40% of the cellular energy for their biogenesis (1). Ribosome biogenesis is a multistep process involving a coordinated network of RNA–RNA, RNA–protein, and protein–protein interactions. The process begins with the transcription of rRNA, its processing, nucleoside modifications, structural rearrangements, synthesis, and interaction of ribosomal proteins (r-proteins) (2). In cells, RNA processing enzymes, modification systems, and chaperone-like factors facilitate ribosome biogenesis. Any deficiencies in this process may produce ribosomes that fail to maintain the fidelity of protein synthesis and cause cellular toxicity/death (3). Deficiencies in biogenesis factors impart cold sensitivity to Escherichia coli and affect bacterial drug resistance and virulence. In humans, genetic defects resulting in imperfect ribosome biogenesis are the cause of various ribosomopathies (4–7). Thus, a better understanding of ribosome biogenesis is a fundamental requirement to develop antimicrobials and various therapies (8–12).

Of the two ribosomal subunits in E. coli, the small subunit contains a 1,542 nucleotide long rRNA (16S rRNA) along with 21 r-proteins, whereas the large subunit contains two rRNAs of 2,904 (23S rRNA) and 120 (5S rRNA) nucleotide lengths along with 33 r-proteins. The three rRNAs are synthesized as parts of a single transcript and the assembly of r-proteins begins cotranscriptionally. As the assembly advances, the precursor rRNA is cleaved by RNase III into immature units of 16S rRNA (as 17S), 23S rRNA (as 25S), and 5S rRNA (as 9S) rRNAs. The 17S rRNA retains unprocessed extensions of 110 and 33 nucleotides on the 5′ and 3′ ends, respectively. Trimming of the extensions by RNase G and other RNases matures 16S rRNA. Incorporation of r-proteins occurs in three stages. The primary r-proteins interact directly with the rRNA, followed by the secondary r-proteins requiring one or many primary r-proteins, and finally the tertiary r-proteins, which are the late proteins to be incorporated (13–16).

In vitro reconstitutions suggested that ribosomes could self-assemble from the mature rRNAs and r-proteins (15, 17, 18). However, the in vitro assembly requires nonphysiological conditions of high temperatures and salt concentrations (19). Besides, in vivo assembly is coupled with rRNA transcription, allowing each domain to fold before the next domain is transcribed, and occurs in the presence of the flanking sequences in the immature 16S rRNA. Thus, in vitro studies do not recapitulate in vivo events of coordination between the transcription, assembly, and processing of pre-rRNAs (2).

As noted in bacteria, a large number of biogenesis/assembly factors and modification enzymes guide ribosome assembly (20). Deficiencies in biogenesis factors, or the assembly process due to stress or otherwise, result in accumulation of ribosomes with immature rRNA ends (21, 22). Whereas the unprocessed flanks at the 5′ and 3′ ends of rRNAs negatively affect the assembly process, they are also important in quality control of the assembled ribosomes, and are cleaved only in the late stages of assembly. The mechanism of how ribosome assembly is completed by final trimming of the rRNA flanks by RNases has remained unclear. In this study, a genetic observation in E. coli has revealed a role of initiator tRNA (i-tRNA) as a biogenesis factor in the final stages of ribosome assembly.

Results

Mutations in the Anticodon Stem of i-tRNA Impact 16S rRNA Processing.

E. coli possesses four i-tRNA genes, three at the metZWV locus and the fourth one at the metY locus. Earlier, we showed that deletion of metZWV (encoding ∼75% of total i-tRNA) confers cold sensitivity to E. coli (23–25). Whereas the ΔmetZWV strain (Fig. 1 A, i) does not show a significant growth defect at 37 °C, its growth is compromised at 22 °C (Fig. 1 A, ii). Cold sensitivity is often a consequence of a ribosome biogenesis defect (2) and results in accumulation of precursor rRNAs. To investigate for a possible connection between i-tRNA and ribosome biogenesis, we carried out Northern blotting of total RNA using DNA oligomers that anneal to the body of 16S rRNA or exclusively to its unprocessed precursor regions (p16S 3′ and p16S 5′). Compared with the WT strain (KL16), we observed increased accumulation of precursor 16S rRNA (p16S) at both the 5′ and the 3′ ends in ΔmetZWV strain at 22 °C (Fig. 1 A, iii and iv, compare lane/bar 4 with 3). In fact, in ΔmetZWV, even at 37 °C there was a small increase in p16S rRNA (lanes/bars 1 and 2).

Fig. 1.

Effect of i-tRNA on ribosome biogenesis. (A) Depletion of i-tRNA confers cold sensitivity. (i) Schematic depiction of E. coli KL16 strains for their full complement of four i-tRNA genes (WT, indicated as wt), or deleted for three (metZWV) of the four i-tRNA genes (ΔmetZWV). (ii) Plate assay showing growth of WT and ΔmetZWV strains at 37 °C and 22 °C. Overnight cultures were streaked on LB agar plates and incubated at the indicated temperatures and monitored for the growth. (iii) Analysis of immature 16S rRNA. Total RNA was prepared from strains grown at 37 °C and 22 °C and Northern analysis was performed with DNA oligomers against mature rRNA (16S probe), 5′- or 3′-immature regions of 16S rRNA (p16S 5′ and p16S 3′). (iv) Quantification of relative changes in precursor rRNA (p16S) at 5′ and 3′ ends relative to WT at 37 °C (lane 1) is shown as bar diagram. (B) Overexpression of i-tRNA_3GC is toxic to ΔmetZWV strain. (i) Schematic depiction of WT and ΔmetZWV strains, and the plasmids (vector, pmetY, and pmetY_3GC) used to transform them. The anticodon stem loop of i-tRNAs indicating the regions of difference between the i-tRNA and i-tRNA_3GC are shown above the pmetY and pmetY_3GC. (ii) Plate assay for growth of WT (harboring empty vector) and ΔmetZWV strains harboring pmetY_3GC (i-tRNA_3GC) or pmetY (i-tRNA) at 37 °C and 22 °C. (iii) Analysis of precursor 16S rRNA (p16S) from strains grown at 37 °C or incubated at 22 °C for 3 h after growing at 37 °C by Northern analysis. (iv) Quantification of fold changes in immature rRNA level at the 5′ or 3′ ends is shown as bar diagram. (C) Overexpression of i-tRNA_3GC confers cold sensitivity to WT E. coli. (i) Schematic depiction of WT and ΔmetZWV strains and the plasmids (vector, pmetY, and pmetY_3GC) used to transform them as in B. (ii) Plate assay showing growth of WT (KL16) transformed with vector alone, pmetY_3GC, or pmetY. (iii) Analysis of immature 16S rRNA from strains grown at 37 °C and 22 °C by Northern analysis. (iv) Quantification of fold change in immature 16S rRNA at 5′ and 3′ ends.

Introduction of the pmetY plasmid encoding i-tRNA into the ΔmetZWV strain (Fig. 1 B, i) rescued both the cold sensitivity and the 16S rRNA processing defects, suggesting these to be specific effects of i-tRNA deficiency (Fig. 1 B, ii, compare sectors 3 and 4 with 7 and 8 at 22 °C; and B, iv, bar 6 with 8). To better understand this phenomenon, we focused on the single most conserved feature (across all domains of life) of the presence of three consecutive GC base pairs (GC/GC/GC, or 3GC pairs) in the anticodon stem of i-tRNAs. Interestingly, when we introduced pmetY_3GC (encoding i-tRNA_3GC, wherein the 3GC pairs were mutated to UA/CG/AU, Fig. 1 B, i) into the ΔmetZWV strain, it conferred toxicity at both the growth temperatures of 37 °C and 22 °C (Fig. 1 B, ii, compare sectors 5 and 6 with 3 and 4), and a notable increase in unprocessed 16S rRNA (Fig. 1 B, iii and iv, lanes/bars 3 and 7). In fact, in the ΔmetZWV strain, toxicity of i-tRNA_3GC was so severe that it often resulted in isolation of suppressors with increased levels of i-tRNA (SI Appendix, Fig. S1), making the combination of i-tRNA_3GC in the ΔmetZWV strain intractable for further investigation. Interestingly, however, introduction of pmetY_3GC in the KL16 strain (Fig. 1 C, i) WT for its i-tRNA genes (metZWV and metY) also showed cold sensitivity at 22 °C (Fig. 1 C, ii, compare sectors 3 and 4 with 1 and 2) albeit to a lesser extent than the ΔmetZWV strain (Fig. 1B). More importantly, as the toxicity was not seen at 37 °C (Fig. 1 C, ii, compare sectors 1 and 2 with 3 and 4; SI Appendix, Fig. S2A), it provided us with a steadfast system to investigate the phenomenon of i-tRNA_3GC-mediated cold sensitivity. Northern blotting revealed an increased level of immature rRNAs when i-tRNA_3GC was overexpressed at 22 °C (Fig. 1 C, iii and iv, compare lane/bar 5 with 4). As there was no significant accumulation of precursor 23S rRNAs (SI Appendix, Fig. S2B), the role of the 3GC pairs was more specific to 16S rRNA processing. At 22 °C even the WT strain showed some biogenesis defect (approximately twofold) (Fig. 1 C, iv, compare bar 4 with 1), which could be partially rescued by increased i-tRNA (pmetY, lanes 3 and 6).

For further analysis of the requirement of individual GC pairs in 16S rRNA maturation, we used various mutants of 3GC base pairs and observed that a full complement of the 3GC pairs is required for efficient ribosome biogenesis (SI Appendix, Fig. S3). We then used i-tRNA mutants with altered anticodons (CAU mutated to GAC, GAU and CUA in pmetY_GAC, pmetY_GAU, and pmetY_CUA, respectively) and their derivatives with 3GC mutations (pmetY_GAC/3GC, pmetY_GAU/3GC, and pmetY_CUA/3GC). Unlike pmetY_3GC, neither the anticodon mutants nor their 3GC derivatives caused cold sensitivity (SI Appendix, Fig. S4). These observations suggest that for i-tRNA_3GC to phenocopy i-tRNA depletion (Fig. 1A), requirement of the WT anticodon is vital. A likely step at which i-tRNA_3GC competed with i-tRNA could be the stage of initiation complex (IC) formation. This would also explain severe toxicity of i-tRNA_3GC in the ΔmetZWV strain (Fig. 1B).

The i-tRNA_3GC in 70S Interferes with Ribosome Biogenesis.

To further check for the role of 3GC pairs in ribosome biogenesis, KL16 strains (WT) harboring vector alone, pmetY_3GC, or pmetY were grown at 22 °C for polysome profiles (Fig. 2A). The status of 16S rRNA was analyzed in the 30S, 50S, 70S, and polysome fractions by Northern blotting (Fig. 2B). The relative analysis of immature to total 16S rRNA showed that i-tRNA_3GC caused significant accumulation of immature 16S rRNA lacking final 3′ end processing in the 70S fraction (Fig. 2 C, i, compare bar 7 with 3). Although i-tRNA_3GC overexpression caused a defect even at the 5′ end processing (Fig. 1 C, iii and iv), this species of precursor 16S rRNA did not accumulate in 70S (Fig. 2 C, ii, compare bar 7 with 3). Further, in agreement with the growth phenotype (Fig. 1 C, ii), cultures grown at 37 °C did not result in any significant increase in precursor 16S rRNA (SI Appendix, Fig. S5). More importantly, an increased level of i-tRNA_3GC in 70S ribosome at 22 °C (SI Appendix, Fig. S6, i and ii, compare lanes/bars 3 and 4 with 7 and 8) indicated its impact on 16S rRNA processing mainly at the 70S ribosome level. As the 16S rRNA maturation defect we detected in 70S ribosome was for 3′ end processing, in the subsequent experiments, we monitored mainly for the 16S rRNA 3′ end processing.

Fig. 2.

Effect of i-tRNA_3GC on maturation in 70S. (A) Analysis of ribosomal profiles upon overexpression of i-tRNA_3GC or i-tRNA. KL16 strain (WT) transformed with empty vector, pmetY_3GC or pmetY was grown at 22 °C and ribosomes were separated on 15–35% sucrose density gradient and analyzed. (B) The RNAs from fractions were isolated using hot phenol, separated on 1.2% formaldehyde agarose gels, transferred onto Nytran membranes, and probed for 16S rRNA and 16S rRNA precursors using 16S, p16S 3′ and p16S 5′ probes. (C) Quantification of relative changes in the precursor forms of 16S rRNA.

Genetic Interactions with Other Biogenesis Factors.

Is the role of i-tRNA in ribosome biogenesis a direct one or an indirect effect due to deficient translation of some ribosome biogenesis factors? An earlier quantitative analysis of the whole cell proteome had suggested that in the ΔmetZWV strain, there are no significant effects in the synthesis of biogenesis factors or r-proteins (25). When we checked for translation deficiencies by deep sequencing of the mRNAs (RNA sequencing) bound to 70S ribosomes (SI Appendix, Fig. S7), we found no defects in translation of mRNAs of the known ribosome biogenesis factors (19). Furthermore, when we overexpressed the known and the putative biogenesis factors in the ΔmetZWV strain (SI Appendix, Fig. S8) we found no significant changes in the ΔmetZWV strain. These results strongly suggest the direct function of i-tRNA as a biogenesis factor.

The C-terminal tail of S9 r-protein interacts with i-tRNA (26) and the lack of three amino acids in the S9 tail (S9Δ3) confers cold sensitivity (27). Overexpression of i-tRNA_3GC in this background showed enhanced toxicity even at 37 °C, and the strain was unable to grow at 22 °C (Fig. 3 A, compare curves 1 and 2 of A, ii with A, i, both at 37 °C and 22 °C). Overexpression of i-tRNA rescued the growth defects (compare curves 1 and 3 of A, ii). The precursor 16S rRNA showed an increase with overexpression of i-tRNA_3GC (Fig. 3 B, i and ii, compare lane/bar 2 with 1) and decrease with overexpression of i-tRNA (compare lane/bar 3 with 1).

Fig. 3.

Genetic interactions of i-tRNA with other biogenesis factors. (A) Growth of WT (KL16), S9Δ3, ΔrsmB+D, and ΔrbfA strains harboring empty vector, pmetY_3GC, or pmetY at 37 °C and 22 °C. (B) Analysis of immature 16S rRNA 3′ end in total RNAs from the same strains mentioned above (i). Quantification of relative changes in immature 16S rRNA 3′ with respect to WT (bar 10) at 22 °C (ii).

Another important feature in the P site is the methylations at 966 and 967 positions of 16S rRNA by RsmD and RsmB, respectively, whose deficiencies compromise ribosome biogenesis (28). Overexpression of i-tRNA_3GC in RsmB- and RsmD-deficient strains (ΔrsmBΔrsmD, ΔrsmB+D) showed heightened toxicity (Fig. 3 A, iii, compare curves 1 and 2), and accumulation of immature 16S rRNA (Fig. 3 B, i and ii, compare lane/bar 5 with 4). Both defects could be rescued by i-tRNA overexpression [compare curves 1 and 3 (Fig. 3A) and lanes/bars 4 and 6 (Fig. 3B)]. Interestingly, lack of 966 and 967 position methylations is known to compromise i-tRNA binding (28).

We then checked for the genetic interaction of i-tRNA with RbfA, a well-known ribosome biogenesis factor (mainly the 5′ domain of 16S rRNA) in E. coli (20, 21). RbfA deficiency caused a large accumulation of immature rRNAs at lower growth temperatures (Fig. 3 B, i and ii, compare lanes/bars 7 and 10). Overexpression of i-tRNA_3GC in the ΔrbfA strain heightened neither the retarded strain growth (Fig. 3 A, iv, curves 1 and 2) nor the immature 16S rRNA levels (Fig. 3 B, i and ii, compare lanes/bars 7 and 8). Overexpression of i-tRNA rescued neither the cold sensitivity nor the biogenesis defect, suggesting that i-tRNA functioned downstream of RbfA. Likewise, i-tRNA overexpression in strains deficient for RsgA, RimO, RimM, or KsgA did not improve the growth of these strains nor did these deficiencies show any synthetic toxicities with the expression of i-tRNA_3GC (over the control) (SI Appendix, Fig. S9). Further, overexpression of S1 diminished the effect of i-tRNA_3GC on both the cold sensitivity as well as the accumulation of precursor rRNAs (SI Appendix, Fig. S10). Importantly, analysis of such a large number of genetic interactions (Fig. 3 and SI Appendix, Fig. S9) also support a direct role of i-tRNA in ribosome biogenesis.

The 3GC Base Pairs Are Needed for the 3′ End Maturation of 16S rRNA.

Because final maturation of 16S rRNA occurs as the last step of the biogenesis, we checked for genetic interactions between rRNA processing enzymes and i-tRNA. The strains lacking RNases such as RNase II (rnb), RNase R (rnr), RNase PH (rph), RNase D (rnd), RNase T (rnt), RNase G (rng), RNase BN (rbn), and YbeY (ybeY) were transformed with pmetY_3GC and checked for accumulation of 16S rRNA precursor. Overexpression of i-tRNA_3GC in RNase II- and RNase R-deficient strains resulted in a significantly increased accumulation of the precursor 16S rRNA (Fig. 4 A, i and ii, compare lanes/bars 4 and 10 with 2). Deficiencies of other RNases tested, did not result in any significant increase in i-tRNA_3GC-mediated accumulation of precursor 16S rRNA (over the control). Cold-sensitive phenotypes also mimicked a similar trend [Fig. 4B and SI Appendix, Fig. S11A, compare the difference between curves 3 and 4 of the strains with that of control (C)]. Overexpression of i-tRNA_3GC led to increased cold sensitivity in the strains deficient for RNase II and RNase R, which are mainly involved in the 3′ maturation of 16S rRNA (Fig. 4 B, compare curves 3 and 4 of B, ii and B, iii with B, i). No significant interactions were observed with the other RNases tested (SI Appendix, Fig. S11A).

Fig. 4.

Effect of enzymes involved in the maturation of rRNAs. (A) Effect of the absence of RNases involved in rRNA maturation. The WT (control, C) or the deletion strains such as RNase B, D, G, R, T, BN, PH, and YbeY (rnb, rnd, rng, rnr, rnt, rbn, rph, and ybeY, respectively) were transformed with either empty vector (−) or pmetY_3GC (+). Total RNA from cultures grown at 22 °C was analyzed for immature 16S rRNA by Northern blotting (i). Quantification of the relative changes in the immature region of 16S rRNA relative to total 16S rRNA. The fold changes with respect to vector control were shown for individual backgrounds (ii). (B) Growth curves showing the effect of i-tRNA_3GC in WT (C) parent (i), Δrnb (ii), and Δrnr (iii) strains at 37 °C and 22 °C. (C) Effect of overexpression of RNase PH, RNase R, and YbeY. Analysis of growth by plate assay of KL16 harboring vector or pmetY_3GC (i-tRNA_3GC) along with overexpression of rph, rnr, or ybeY from compatible plasmids (prph, prnr, or pybeY) at 37 °C and 22 °C. Analysis of immature 16S rRNA by Northern blotting (ii) and the relative changes (iii).

RNase II, RNase PH, PNPase, and RNase R are involved in 16S rRNA 3′ end maturation (29). However, we did not observe a strong effect of rph deletion in the i-tRNA_3GC background (SI Appendix, Fig. S11 A, vi). It may be noted that an already existing mutation in the rph gene in E. coli BW25113 results in a C-terminally truncated and less active RNase PH (30). Hence, rph deletion in the BW25113 (i.e., in the background already deficient for RNase PH) is not expected to cause a major impact over the strain background (SI Appendix, Fig. S11 A, compare A, i with A, vi). However, we typically see that i-tRNA_3GC overexpression causes increased cold sensitivity in the BW25113 strain over the KL16 strain (with WT rph), supporting the genetic interaction (SI Appendix, compare Fig. S2 A, ii and Fig. S11 A, i). This finding suggests that the 3′ maturation defect due to the lack of RNase PH, RNase R, or RNase II enhances the ribosome maturation defect with overexpression of 3GC mutant i-tRNA.

To further our understanding of the defect in 16S rRNA processing, we overexpressed the 3′ end processing enzymes, such as RNase PH and RNase R, from compatible plasmids in i-tRNA_3GC overexpression backgrounds (Fig. 4C and SI Appendix, Fig. S11B). Interestingly, overexpression of just the 3′-end processing enzymes rescued the cold sensitivity of the strains (Fig. 4 C, i, compare sectors 4 and 6 with 2; SI Appendix, Fig. S11 B, compare curves 3 and 4 between B, i and B, iii) and decreased accumulation of precursor rRNA (Fig. 4 C, ii and iii, compare lanes/bars 4 and 6 with 2). Recently, another enzyme YbeY was proposed to be involved in the 3′-end processing, mainly as a quality-control step of degrading 70S ribosomes with immature 30S ribosome (31). Overexpression of YbeY could also rescue the cold sensitivity (Fig. 4 C, i, compare sector 8 with 2; SI Appendix, Fig. S11 B, iv) and lower the level of precursor 16S rRNA (Fig. 4C, compare lane/bar 8 with 2).

Impact of Translation Factors on Ribosome Maturation.

Our observations suggest that initiation complex formation with i-tRNA is needed in maturation of ribosomes. To understand the impact of various initiation factors or recycling factors, we overexpressed them (from compatible plasmids) in WT strains harboring vector control, pmetY_3GC or pmetY (Fig. 5). IF1 (infA) did not majorly impact i-tRNA_3GC-mediated cold sensitivity or 16S rRNA maturation (Fig. 5 A, ii and B, i and ii, compare lane/bar 5 with 2). IF2 (infB) caused severe cold sensitivity, which was partially rescued by i-tRNA (Fig. 5 A, iii, compare curve 3 with 1 at 22 °C). However, because of the severe cold sensitivity caused by IF2, additional impact of i-tRNA_3GC could not be investigated in this background (Fig. 5A, compare curves 1 and 2 of A, iii at 22 °C). And, while i-tRNA lowered accumulation of immature 16S rRNA, impact of i-tRNA_3GC could not be discerned (Fig. 5 B, i and ii, lanes/bars 7–9). Overexpression of IF3 (antiassociation factor) caused toxicity with i-tRNA_3GC at both 37 °C and 22 °C (Fig. 5 A, iv, compare curve 2 with 1), and an increased accumulation of 16S rRNA precursor (Fig. 5 B, i and ii, lanes/bars 10 and 11; compare with lanes/bars 1 and 2).

Fig. 5.

Effect of overexpression of translation factors in ribosome biogenesis. (A) E. coli KL16 harboring vector contol, pmetY_3GC, or pmetY were transformed with the compatible empty vector control (C) (i), pinfA (ii), pinfB (iii), pinfC (iv), pfrr (v), or pfrr-infC (vi) and the growth was monitored at 37 °C and 22 °C in LB Amp–Tet. Similar analysis was carried out in the infC135 strain (vii). (B) Levels of precursor 16S rRNAs were analyed by Northern blotting (i), and fold differences of p16S 3′ were quantified (ii).

Ribosome recycling factor (RRF), which dissociates the 70S ribosomal complexes to recycle posttermination and stalled ribosomes, has been implicated in initiation (32), and the RRF function is assisted by IF3 (33). Overexpression of RRF or RRF-IF3 enhanced both the cold sensitivity (Fig. 5 A, v and vi) and precursor 16S rRNA accumulation caused by i-tRNA_3GC (Fig. 5 B, i and ii, lanes/bars 13 and 14 and 16 and 17, compare with lanes/bars 1 and 2). Further, in a strain having the mutant IF3 (infC135) allele, overexpression of i-tRNA_3GC did not enhance cold sensitivity (Fig. 5 A, vii) or accumulation of precursor 16S rRNA (Fig. 5 B, i and ii, compare lanes/bars 19 and 20). E. coli LJ14, possessing a temperature-sensitive allele of frr (encoding RRF) showed diminished cold sensitivity at 22 °C (SI Appendix, Fig. S12, i) and lowered accumulation of immature rRNA, upon i-tRNA_3GC overexpression (SI Appendix, Fig. S12, ii and iii, compare lanes/bars 1 and 2, and 4 and 5).

Effect of Protein Synthesis Inhibitors on 16S rRNA Processing.

To address whether translation per se is involved in 16S rRNA processing, we treated E. coli KL16 with various antibiotics and analyzed the status of 16S rRNA. As shown in SI Appendix, Fig. S13, treatment with the antibiotics led to increase in accumulation of 16S rRNA precursor. Biogenesis defects upon treatment with translation inhibitors were also seen earlier (34, 35). It was proposed that an imbalance between rRNA transcription and sufficiency of r-proteins due to translational inhibition led to the defect. Thus, to monitor processing of the available rRNA transcripts, we first inhibited new RNA synthesis by treating the cells with rifampicin and then followed maturation of the available rRNA upon addition of the inhibitors (Fig. 6A). Rifampicin treatment alone led to a gradual decrease in immature rRNA accumulation at 15 and 30 min (Fig. 6 A, i, compare −Rif lane 1 with +Rif lane 9; and Fig. 6 A, ii, compare bars 1 and 2 with 3 and 4). Chloramphenicol (Cm), kasugamycin (Kas), or spectinomycin (Spectino) did not affect the maturation process, although they increased the immature rRNA levels in the absence of rifampicin (Fig. 6 A, i and ii, compare −Rif with the respective +Rif samples). Interestingly, tetracycline (Tet) that, in addition to binding in the A site, inhibits i-tRNA binding in the P site (36), prevented the maturation completely (Fig. 6 A, ii, compare bars 1–4 with 13–16). Further, overexpression of i-tRNA_3GC (pmetY_3GC) or depletion of i-tRNA (ΔmetZWV) resulted in enhanced accumulation of immature 16S rRNA under all treatments (SI Appendix, Fig. S14). Conversely, there was increased processing of 16S rRNA upon i-tRNA overexpression (SI Appendix, Fig. S14). These observations suggest that a full translation cycle is not needed for efficient 16S rRNA processing by i-tRNA.

Fig. 6.

Requirement of IC in 16S rRNA maturation. (A) Impact of translational inhibitors on 16S rRNA processing. Total RNA was separated on formamide agarose gel and probed for total 16S rRNA and 16S precursor rRNA (i) and quantified for 16S rRNA processing (ii). (B) Effect of lamotrigine on ribosome biogenesis. The growth curve of KL16 with vector control, or pmetY_3GC (i-tRNA_3GC) or ΔmetZWV with vector control or pmetY (i-tRNA) complementation were analyzed in presence of increasing concentrations of lamotrigine at 22 °C (i) as well as 37 °C (ii).

Recently, it was reported that lamotrigine, which binds IF2, led to defective assembly of the ribosomal subunits (37). The mechanism of lamotrigine-mediated inhibition of ribosome biogenesis is unclear. Thus, to check for any interactions between i-tRNA and lamotrigine, we checked the effect of i-tRNA_3GC overexpression or i-tRNA depletion (ΔmetZWV strain). Both conditions led to enhanced cold sensitivity (Fig. 6 B, i, compare curves 2 and 3 with 1), although the growth at 37 °C was not significantly impacted (Fig. 6 B, ii). The i-tRNA overexpression rescued the biogenesis defect (Fig. 6 B, i, compare curve 4 with 1 of 40 μM lamotrigine), suggesting that the effect of lamotrigine is also mediated via interference of the initiation pathway. Such an effect was not observed with other antibiotics (SI Appendix, Fig. S15). Rescue of lamotrigine-mediated cold sensitivity by i-tRNA, and the fact that both lamotrigine and i-tRNA bind to IF2, further support our finding of the role of i-tRNA in ribosome biogenesis at the step of initiation.

Discussion

Recently, we showed that the hallmark feature of the 3GC pairs in the anticodon stems of i-tRNAs is crucial in transiting it from 30S IC to elongation-competent 70S (38). The present observation that mutations in 3GC pairs result in cold sensitivity and accumulation of immature 16S rRNA in E. coli has revealed yet another important role of this (3GC pairs) evolutionarily conserved feature, in ribosome biogenesis.

Our observations in Fig. 4 show that the genetic interactions between i-tRNA and the RNases (RNase II, RNase R, and RNase PH) are important in 16S rRNA 3′-end processing. The antibiotics blocking i-tRNA binding in the P site interfere with the final processing of the 16S rRNA (Fig. 6). Further, the observation that i-tRNA_3GC with WT anticodon but not the mutant anticodons (even though they are aminoacylated and formylated) are inhibitory also supports that the processing is triggered by IC formation (SI Appendix, Fig. S4). However, does the maturation occur in 30S IC or at the 70S level? The analysis of the ribosome profile at 22 °C (Fig. 2 and SI Appendix, Fig. S6) showed accumulation of unprocessed 16S rRNA (together with i-tRNA_3GC) in 70S, indicating that the processing occurred at the 70S stage. Interestingly, overexpression of IF2 alone also enhanced the biogenesis defect. At its high cellular levels, IF2 may target noninitiator tRNAs into 70S and impede 16S rRNA processing. Increased toxicity by overexpression of IF3 (antiassociation factor) and/or RRF (dissociates 70S) suggests that a productive 70S may be needed for 16S rRNA maturation. The mutant IF3 (infC135) that allows initiation to occur with i-tRNA_3GC rescued the biogenesis defect. Furthermore, the strain temperature sensitive for RRF, also rescued i-tRNA_3GC-mediated cold sensitivity (and lowered accumulation of precursor 16S rRNA compared with vector control; SI Appendix, Fig. S12), further suggesting that much of the final 16S rRNA processing occurred in the 70S complex.

Recent studies in eukaryotes suggest that the maturing ribosomes undergo one round of subunit association as a quality check (39). Eukaryotes use eIF5B, an ortholog of bacterial IF2, to promote the association of pre-40S and -60S subunits to form an 80S subunit, before maturation of 20S rRNA to 18S rRNA, but that this occurs in an i-tRNA- and mRNA-independent manner (39, 40). In bacteria, 70S complex formation is mediated by IF2, requiring i-tRNA for stable binding. Thus, formation of 70S might be required for ribosome biogenesis/quality control in bacteria. This possibility is further supported by the effect of lamotrigine, which binds IF2 in its domain II (37). As this domain is shown to be important for binding to 30S and 50S subunits and (41), as in our experiments, i-tRNA overexpression slightly rescues lamotrigine-mediated cold sensitivity, we suggest that the effect of lamotrigine on ribosome biogenesis is due to decreased 70S formation. Taken together, our observations provide compelling evidence for the role of i-tRNA in the final maturation of 16S rRNA at the level of the 70S complex (Fig. 7). In such a scenario, lack of efficient 50S maturation might also impact 30S ribosome maturation (by affecting the 70S complex formation). Thus, our proposal of requirement of 70S complex for a final maturation of 16S rRNA precursor might also explain the unanswered genetic observations from the 50S biogenesis factors, which were shown to affect 30S ribosome maturation (2).

Fig. 7.

Schematic of i-tRNA participation in ribosome maturation at the 70S stage. After the assembly of the 30S subunit, immature 30S possessing precursor 16S rRNA form 70S IC with the help of i-tRNA as well as 50S. During the complex formation, i-tRNA (guided by its 3GC base pairs) interacts with P-site elements, which in turn facilitates RNase-mediated processing of the 16S rRNA extensions. Evidence presented in this study suggests that the final processing occurs in the 70S complex.

However, how does i-tRNA, especially its 3GC pairs, function as ribosome biogenesis factor? Overexpression of i-tRNA rescues the biogenesis defect caused either by the lack of 16S rRNA methylations in the P site (positions 966 and 967) or the C-terminal tail residues of S9 that affect i-tRNA binding (28, 42). The i-tRNA could serve as a “template” for proper structuring of the P-site elements (particularly the rRNA) by establishing contacts via its 3GC pairs, and serve as a signal for final processing. Alternatively, the overall conformational changes that occur in the ribosome during the initiation pathway might themselves signal final processing. The duplex between the SD sequence in mRNA and aSD sequence in the 3′-terminal of 16S rRNA, within the 70S complex, may also serve to limit the single strand-specific 3′-to-5′ exonuclease activity of RNase R beyond the precursor sequences (Fig. 7). Interestingly, the 3GC pairs, as well as the mRNA interactions, are known to affect the movements around the region of h28 in 16S rRNA (43). Binding of i-tRNA may affect the 16S rRNA 3′-end region through the conserved residues (G1338 and A1339 that interact with the 3GC pairs). Thus, unique interactions occurring between the i-tRNA and the 16S rRNA conserved residues at the 70S stage might be guiding the final maturation of 16S rRNA.

YbeY has been shown to degrade 16S rRNA from the 3′ end, but at the 70S stage (31). This enzyme is mainly attributed to the degradation of defective ribosomes as quality control. If both final maturation and quality control (YbeY-mediated degradation) happen at the 70S stage, how are the two processes discriminated? We believe that the maturation occurs at an early stage of 70S formation. If the maturation fails, the ribosomes may be targeted for degradation at the later stages in 70S. This notion is further supported by the fact that, except tetracycline, none of the other translation inhibitors prevented 16S rRNA maturation. Most likely, only the initiation step (and not the entire translation cycle) is crucial for the maturation process. Overall the present study provides compelling evidence that the i-tRNA plays a crucial role in the final steps of maturation of the 30S ribosome.

Materials and Methods

Bacterial Strains, Plasmids, DNA Oligomers, and Growth and Culturing Conditions.

The bacterial strains, plasmids, and the DNA oligomers are listed in SI Appendix, Tables S1–S3, respectively. E. coli KL16, BW25113, and their derivatives were grown in LB liquid or LB–agar plates containing 1.5% bacto-agar (Difco). Unless mentioned otherwise, media were supplemented with ampicillin (Amp, 100 µg/mL), Cm (30 µg/mL), kanamycin (Kan, 25 µg/mL), or Tet (7.5 µg/mL) when required.

Bacterial Growth Analysis.

Bacterial growth was monitored by plate assays and/or growth curves. For plate assays, overnight cultures were streaked on desired agar plates and incubated at desired temperatures for various time intervals. For growth curve analysis, cultures (four replicates/colonies of each strain) were grown in LB with the desired antibiotic(s) until they reached saturation at 37 °C. A 200-μL volume of a 10⁻² to 10⁻³ dilution of the cultures in the required medium was taken in honeycomb plates and placed in an automated Bioscreen C growth reader. OD₆₀₀ was measured every hour. Mean OD values and SDs were calculated and the data were plotted with time on the x axis against OD₆₀₀ on the y axis using GraphPad Prism software.

Analysis of Total RNA.

Total RNA from 2 mL midlog-phase cultures were isolated using the hot phenol method. Briefly, cell pellets were resuspended in 300 μL lysis solution (30 mM Tris⋅HCl pH 8.0, 0.1 M NaCl, 5 mM Na₂ EDTA, 1% SDS, 4 mM β-mercaptoethanol) and mixed with an equal volume of hot phenol (saturated with sodium acetate, pH 4.5, heated at 65 °C) and incubated at 65 °C for 10 min. An equal volume of chloroform was added and the two layers were separated. RNA were recovered from the aqueous phase followed by isopropanol precipitation and taken up in 50 μL buffer (0.1 mM sodium acetate, pH 4.5 and 0.1 mM Na₂ EDTA). Total RNA (∼10 μg) was separated on 1.2% formaldehyde agarose gel and transferred onto Nytran membrane (44).

Polysome Profiling.

The total ribosomal preparations from E. coli were carried out as before (38). Translation was inhibited by 100 μg/mL Cm before harvesting cells and chilled on an ice–salt mix. Approximately 10–20 OD₂₆₀ of preparation was loaded on 15–35% (wt/vol) sucrose gradients (buffered in 20 mM Hepes-KOH, pH 7.5, 50 mM NH₄Cl, 10 mM MgCl₂, 4 mM β-mercaptoethanol) prepared using a BioComp gradient master (BioComp Instruments) in polyclear tubes and separated using an SW55 rotor at 45,000 rpm for 2.5 h at 4 °C. Gradients were fractionated at a flow rate of 0.3 mm per second using a BioComp gradient fractionator and subunits were manually collected by monitoring the absorption at 254 nm using a BIO-RAD Econo UV monitor.

Analysis of tRNA.

tRNA from various fractions of polysome profiles were extracted using the hot phenol (saturated with sodium acetate, pH 4.5) and chloroform treatment, followed by precipitation using isopropanol. The tRNAs were separated on native PAGE and transferred on to Nytran membrane as described earlier (38).

Northern Blotting and Estimation of Relative Changes.

RNAs were fixed onto the Nytran membrane by UV cross-linking at 120 mJ/cm² (CL1000 UV Products). The membranes were blocked using prehybridization buffer containing yeast RNA and Denhardt’s solution (1% BSA, 1% Ficoll, 1% polyvinylpyrrolidone 40). The Northern blot analysis was performed using 5′-³²P end-labeled DNA oligomers. For analyzing i-tRNA, Met33 probe and for i-tRNA_3GC, 3GC Rp were used. For analyzing precursor 16S rRNAs p16S 3′- and 5′-specific probes, and for total 16S rRNA, the 16S probe against the mature region were used (SI Appendix, Table S3). The same blots were probed sequentially by p16S 3′, 16S rRNA, and finally with the p16S 5′ probe. Blots were exposed to phosphorimager screen and analyzed using the BioImage Analyzer (FLA5100, Fuji Film). Before reprobing of the blot, denaturation (“stripping”) of the previous probe was ensured by reexposure to the Bioimage Analyzer. To calculate relative fold changes, the pixel values in the bands corresponding to the immature rRNA band (5′ or 3′) were divided by the corresponding 16S rRNA band value for each sample and expressed relative to the control sample (which was normalized to unit value).

Ribosome Maturation Assay.

Cells were grown to an OD₆₀₀ of ∼0.6 at 37 °C and either untreated or treated with rifampicin (100 µg/mL) for 3 min and then without or with translation inhibitors (Kas, Spectino, Tet, streptomycin, Cm, fusidic acid, or puromycin) at a concentration of 100 µg/mL, and aliquots were taken after 15 and 30 min. Total RNA was prepared, separated on formamide agarose gel, and probed for total 16S and 16S precursor rRNA.