Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding

Abstract

In prokaryotic systems, the initiation phase of protein synthesis is governed by the presence of initiation factors that guide the transition of the small ribosomal subunit (30S) from an unlocked preinitiation complex (30S preIC) to a locked initiation complex (30SIC) upon the formation of a correct codon-anticodon interaction in the peptidyl (P) site. Biochemical and structural characterization of GE81112, a translational inhibitor specific for the initiation phase, indicates that the main mechanism of action of this antibiotic is to prevent P-site decoding by stabilizing the anticodon stem loop of the initiator tRNA in a distorted conformation. This distortion stalls initiation in the unlocked 30S preIC state characterized by tighter IF3 binding and a reduced association rate for the 50S subunit. At the structural level we observe that in the presence of GE81112 the h44/h45/h24a interface, which is part of the IF3 binding site and forms ribosomal intersubunit bridges, preferentially adopts a disengaged conformation. Accordingly, the findings reveal that the dynamic equilibrium between the disengaged and engaged conformations of the h44/h45/h24a interface regulates the progression of protein synthesis, acting as a molecular switch that senses and couples the 30S P-site decoding step of translation initiation to the transition from an unlocked preIC to a locked 30SIC state.

Keywords: GE81112; X-ray crystallography; antibiotics; protein synthesis; ribosome.

Fig. S1.

Structure of GE81112. Structure of variant B of GE81112 (658 Da) as determined by NMR spectroscopy. The molecule consists of four amino acids: 3-hydroxypipecolic acid, 2-amino-5-[(aminocarbonyl)oxy]-4-hydroxypentanoic acid, 5-amino-histidine, and 5-chloro-2-imidazolylserine (10).

Fig. 1.

GE81112 binds in the ribosomal P site and distorts the ASL mimic. (A) An overview showing the binding site of GE81112 on the 30S subunit with the relevant 16S rRNA helices labeled. (Inset) Two symmetry-related 30S subunits are packed in the crystal such that h6 (blue) of one 30S subunit (red) inserts into the P site of a second subunit (gray) to mimic a P-tRNA. h6 in both subunits is circled. (B) Two orthogonal views of the ASL mimic showing a shift of the backbone between the 30S apo and 30S-GE81112 conformations (red and blue, respectively). Note that U81–U84 of the ASL mimic correspond to U33–U36 in the anticodon loop of the initiator tRNA. The initial unbiased positive Fo-Fc map is contoured at 4 σ (green mesh). (C) The C-terminal tail (K121–R125) of T. thermophilus S13, generally disordered and not visible in 30S crystal structures, becomes structured in the presence of GE81112. The Fo-Fc map is shown. (D) GE81112 is modeled in a binding pocket between S13 and the distorted ASL. (E) Fo-Fc map in the ASL and GE81112 region (Inset) with a zoom-in on the GE81112 electron densities where the ASL density is masked out for clarity. Maps in C and E are rendered at 3 σ and used the bulk solvent modeling protection approach (21).

Fig. S2.

Comparison and alignment of P-site tRNAs and ASL. (A) In 30S crystals h6 of one subunit is inserted into the P-site of a symmetry-related subunit mimicking the ASL of the P-site–bound tRNA. The figure in the panel presents the structure alignment of initiator fMet–tRNA as seen in the 70S structure (PDB ID code 2J00) and the ribosomal spur (h6) as seen in the 30S crystals in the absence of GE81112 (PDB ID code 2ZM6). The structures were aligned using 16S rRNA residues surrounding the P-tRNA. (B) The alignment between the tip of the ASL mimic distorted in the presence of GE81112 with the initiator P-site–bound tRNA as seen in the crystal structure of the 70S ribosome (PDB ID code 2J00) illustrates how the conformational change precludes codon–anticodon interaction. The residues involved in the canonical codon–anticodon interaction are labeled in green (A16–G18) and gray (C34–U36). The distorted ASL mimic (h6) as seen in the presence GE81112 is colored red. The structural alignment was preformed using 16S rRNA elements surrounding the P-tRNA.

Fig. 2.

Overview of conformational changes observed in the 30S subunit in the presence of GE81112. (A) In the GE81112 ribosomal complex, several 16S rRNA residues assume alternative conformations. At the interface of h24a (light blue) and h45 (orange) residues U793, A1519, A1518, G1517, and G1516 are colored red and are drawn in the alternative conformation unique to the GE81112 structure. Similarly residues U81–A85 of the ASL are colored red and drawn as seen only in the presence of GE81112. Note that residues G1491–A1493, although not in an alternative conformation, show significant disorder. The crystallographic refinement indicates that the residues of the ASL, h24a, and h45 that are colored red have occupancies of about 60% and 40%, corresponding to the conformations seen in the presence and absence (PDB ID code 2ZM6) of GE81112. S13 was omitted, because it is not likely to be involved in GE81112 binding in all bacterial species. (B and C) Key residues at the interface of h44/h45/h24a are illustrated in the engaged (B) and disengaged (C) conformation. Although both conformations are observed in the presence of GE81112, the disengaged conformation is predominant in the presence of GE81112, and the engaged conformation is predominant in the apo30S subunits (PDB ID codes 2ZM6 and 1J5E).

Fig. 3.

Inhibition of 30SIC and 70SIC formation by GE81112. (A) Kinetics of 30S preIC formation in the presence (red tracing) and absence (black tracing) of 100 µM GE81112. The ribosomal binding kinetics of fMet–tRNA were followed with a fluorescence stop-flow apparatus making observable the FRET signal generated by the proximity of fMet–tRNA_8-fluo, acting as a donor to IF3₁₆₆-_ALEXA555. (B) Kinetics of locked 30SIC formation in the absence (red trace) or in the presence (black trace) of 1.5 µM GE81112. Binding of f[³⁵S]Met-tRNA to 022 mRNA-programmed 30S subunits to form a 30SIC were measured by rapid nitrocellulose filter binding as described (29). (C) Schematic representation of the 30S preIC complex→30SIC transition. In the 30S preIC complex both mRNA and initiator tRNA are ribosome-bound, but no proper codon–anticodon interaction takes place. A subsequent locking step (inhibited by GE81112) entails P-site codon–anticodon base-pairing that stabilizes the mRNA–initiator tRNA interaction yielding a 30SIC. (D) Stopped-flow kinetics of a 50S subunit docking to 30SIC to produce a 70SIC in the absence (black tracing) or presence (red tracing) of 100 µM GE81112. The association of the 50S subunits with a 30SIC was monitored using the variation in the light-scattering signal as an observable. a.u., arbitrary units.

Fig. S3.

GE81112 does not affect the kinetics of 30S preIC formation. Binding kinetics of fMet–tRNA to the 30S ribosomal subunit in the absence (black tracing) or presence (red tracing) of 100 µM GE81112 measured by a fluorescence stop-flow apparatus making use of two types of observables, namely the FRET signals generated by the proximity of fMet–tRNA_8fluo acting as a donor and IF1_Alexa555 as acceptor (A) and the quenching of IF2_757Alexa488 fluorescence by the approaching fMet–tRNA_8QSY35 (B).

Fig. S4.

Inhibition of translation initiation by GE81112 does not depend on the nature of the initiation codon or the nature of the aminoacyl-tRNA and template. (A) GE81112 concentration-dependent inhibition of [³⁵S]fMet–tRNA binding to 30S ribosomal subunits programmed with 022AUGmRNA (■) or 022AUU mRNA(●) measured by filtration through nitrocellulose filters as described previously (25). (B) In vitro translation of an E. coli cell-free system programmed with 022AUGmRNA (■) or 022AUUmRNA (●). (C) Binding of NAc[³H]–Phe–tRNA to poly(U)-programmed 30S ribosomal subunits to form a pseudoinitiation complex. The reaction was carried out at 5 mM (●) or 15 mM (■) of magnesium acetate (26).

Fig. 4.

Accessibility to hydroxyl radical cleavage of fMet–tRNA in the 30S complexes formed with and without GE81112. (A) Electrophoretic migration of uncleaved (lanes 1 and 2) or hydroxyl radical-cleaved (lanes 3–12) [³²P]fMet–tRNA in the unbound form (lanes 3 and 4), within a 30S complex lacking mRNA (lanes 5 and 6), within a complete 30SIC without GE81112 (lanes 7 and 8), or within a complete 30SIC with increasing amounts (i.e., 1.5, 12, 25, and 50 µM) of GE81112 (lanes 9–12). The L and ΔT1 lanes correspond to the ladders obtained upon alkaline hydrolysis and RNaseT1 digestion of tRNA, respectively. The intensity of the bands obtained upon cleavage in the absence (lanes 7 and 8) or in the presence of 1.5 (black), 12 (blue), 25 (red), and 50 µM (purple) of GE81112 (lanes 9–12) were quantified by densitometry. (B and C) The ratios of the intensities obtained with and without GE81112 are plotted; the anticodon bases are indicated in red. The ratios were calculated using the absolute intensity values (B) or the intensities after subtraction of the averaged intensities of the bands of lanes 1 and 2 considered to represent the spontaneously cleaved background (C). The intensities ratio of U33 could not be calculated. Because of the different extents of the recorded effects, the scales of the ordinate for bases G41–C32 and for G31–G22 are different in both plots. (D) Primer extension analysis of the 002 mRNA sites cleaved by hydroxyl radicals in the presence and absence of GE81112. Uncleaved mRNA (lane 1), mRNA cleaved in the unbound form (lanes 2 and 3) and with a complete 30SIC assembled in the absence (lanes 4 and 5) or in the presence of 0.5 (lanes 6 and 7), 1.5 (lanes 8 and 9), 12 (lanes 10 and 11), 25 (lanes 12 and 13), and 50 µM (lanes 14 and 15) GE81112. Lanes C, T, A, and G are the sequencing ladders. (E) Plot of the ratio of the densitometrically quantified, averaged intensities of bands 6,7/4,5 (black); 8,9/4,5 (blue); 10,11/4,5 (green); 12,13/4,5 (red), and 14,15/4,5 (purple). The SD sequence (green) and the initiation codon AUG (red) are indicated. (F) 3D structure of fMet–tRNA–mRNA interaction (PDB ID code 1YL4) highlighting the tRNA and mRNA regions that are more (red) or less (blue) exposed to cleavage in the presence of GE81112. Further details are given in Materials and Methods.

Fig. S5.

Effect of GE81112 on the rate of exchange between 30S-bound and free IF1 and IF2 and on the kinetics of mRNA binding to the 30S subunit. (A and B) Two types of complexes were prepared in the presence (red tracings) and absence (black tracings) of GE81112. One complex contained fluorescently labeled IF1 _4-ALEXA555 and fluorescein-labeled fMet–tRNA; IF1 dissociation is monitored by the reduction of the FRET signal between the two fluorescent ligands upon the addition of a 10-fold excess of nonfluorescent IF1 (A). The other complex contained fluorescently labeled IF2 _757-Alexa488 and fMet–RNA_8-QSY35 (B). Upon the addition of a 10-fold excess of nonfluorescent factor, the dissociation of IF1 (A) causes a decrease in fluorescence intensity (because of the loss of the FRET signal), whereas IF2 dissociation (B) causes an increase in fluorescence because of diminished quenching. (C and D) Fluorescence stopped-flow binding kinetics of 3′ fluorescein semithiocarbazide-labeled (24) leadered (C) and leaderless (D) mRNA to 30S subunits (black and red tracings) or to 30S subunits containing all components of the initiation complex except for the mRNA (green and blue tracings) in the absence (black and green tracings) or in the presence (red and blue tracings) of 100 µM GE81112.

Fig. S6.

GE81112 alters the mRNA position on the 30S subunit. Primer extension analysis of the 022 mRNA (A) and 003 mRNA (B) sites cleaved by hydroxyl radicals. Uncleaved mRNA (lanes 1 and 2), mRNA cleaved in the unbound form (lanes 3 and 4) and within a complete 30SIC assembled in the absence (lanes 5 and 6) or in the presence of 0.5 µM (lanes 7 and 8), 1.5 µM (lanes 9 and 10), 12 µM (lanes 11 and 12), 25 µM (lanes 13 and14), and 50 µM (lanes 15 and 16) of GE81112. Lanes C, T, A, and G are the sequencing ladders. Bases becoming more exposed in the presence of the antibiotic are indicated by red squares. The positions of the initiation triplet AUG and of the SD sequence (in the case of 022 mRNA) are indicated on the right side of the gels.

Fig. 5.

Ribosomal affinity and dissociation of the IFs in the presence of GE81112. (A) Rate of exchange between fluorescent 30S-bound and nonfluorescent free IF3 in the presence (red tracing) and absence (black tracing) of GE81112. The dissociation of IF3 was determined from the rate of exchange measured by a fluorescence stopped-flow apparatus monitoring the decrease of fluorescence intensity of IF3_166-ALEXA555 upon the addition of a 10-fold excess of nonfluorescent factor. (B–D) Dissociation of the IFs upon association of 30SIC with 50S subunits during the 30SIC→70SIC transition in the presence (red tracing) and absence (black tracing) of GE81112. The dissociation of IF1 (B) and IF3 (D) from 30SIC complexes containing an Alexa 555-labeled factor, and fluorescein-labeled fMet–tRNA was monitored by the decrease in the FRET signal between the two fluorophores. (C) The dissociation of IF2 was monitored by an increase in the fluorescence resulting from the reduced quenching caused by the loss of proximity between IF2_757-ALEXA488 and fMet–RNA_8-QSY35.

Fig. S7.

Effect of GE81112 on the in situ accessibility of 16S rRNA to hydroxyl radical cleavage. Primer extension analysis of the 16S rRNA cleavage sites generated by hydroxyl radicals within the 30S subunit (9) in the presence of increasing concentrations of GE81112 (10–100 μM; increasing concentrations indicated with a triangle). (A and B) Analyses of the 16S rRNA residues 650–710 and 1,340–1,380, respectively. The G and A lanes contain the sequencing reactions. Bases whose accessibility to the cleavage is affected by GE81112 are indicated on the right side of the gels. (C) The positions of 16S rRNA bases becoming more (red) or less (blue) exposed to cleavage in the presence of GE81112 are indicated within the in the 3D 30S structure.

Fig. S8.

Comparison of h44/h45/h24a in the engaged and disengaged conformation. The overall structure of the P-site region encompassing h44 (green), h45 (orange), h24a (cyan), the ASL (blue), and the mRNA (yellow) acquires a different conformation in the presence (A) or absence (B and C) of GE81112. The drug stabilizes the ASL tip in an altered conformation so that the mRNA cannot be positioned properly, and P-site codon–anticodon interaction is hampered. The architecture of the adjacent helices h44, h45, and h24a is altered, reflecting the formation of a different hydrogen bonding network that in the absence of GE8112 involves the mRNA and two additional Mg²⁺ ions (yellow spheres in B and C). In the two complexes, the h44/h45/h24a conformation is either disengaged (A) or engaged (B and C) (also see also Fig. 2 B and C). (C) A close-up and rotated view of B illustrating the potential hydrogen bond network formed by the tip of h44, the mRNA codon, and the ASL. This network could contribute to the stability of the h44 residues that are part of the h44/h45/h24a interface. The mRNA sequence is 5′-AGAAAGGAGGGUUUGGAAUGAACGAGC-3′. The residues most affected by the presence of GE81112 are colored in red (rms value higher than 2 Å) or pink (1 ≤ rmsd < 2 Å).

Fig. S9.

The h44 nucleotides affected by GE81112 take part in the formation of bridges B2a and B3. Representative structure of the subunit interface of the 70S ribosome [PDB ID codes 2J00 (30S, including tRNAs and mRNA) and 2J01 (50S)] is shown in cartoon representation; the mRNA is colored in yellow, the P-site tRNA in blue, H69 and H79 of the 50S subunit in purple, h44 of the 30S subunit in green, and its nucleotides (1406–1409, 1491–1495, and 1417–1419) affected by GE81112 and involved in the switch to the disengaged conformation are in red. These nucleotides are either close to or directly involved in the formation of two intersubunit bridges (B2a and B3) that control and stabilize the interaction between the 30S and 50S subunits. In particular, the conformation of the h44 nucleotides (1409–1410 and 1495–1496) involved in the formation of B2a may have a significant role in the formation of a stable 70SIC, because the tip of H69 on the 50S, which participates in the formation of this bridge, is completely disordered in the free 50S subunit (e.g., PDB ID codes 3CC2 and 2ZJR) (34, 35), but it acquires a stable conformation by induced fit upon binding to the 30S subunit and interaction with h44. In the disengaged conformation, h44 in this region has the highest conformational distortion (see also Fig. 2 and Fig. S8), suggesting that this conformation in the 30SIC would hamper the formation of the bridge B2a, but the formation of this bridge would be favored by the engaged conformation, which is the only one observed in the 70S ribosome, and on the 30S in the presence of a proper accommodation of the P-site ligands. H69 also interacts with the P-site tRNA (blue), further suggesting that in the presence of an incorrect positioning of the mRNA and nonproper codon–anticodon interaction in the P site, the stability of the P-site tRNA would be compromised and possibly explaining why, under these conditions, the fMet tRNA dissociates from the complex. This hypothesis finds support from fMet–tRNA binding experiments in the presence of streptomycin, which induces the disengaged conformation of h44/h45/h24a and causes the release of fMet–tRNA from the 70S ribosome (–39). Similarly, in the absence of methylation of 1518 and 1519, which also causes the stabilization of the disengaged conformation, the association between 30S and 50S is weakened (39).

Fig. 6.

Structural model on the initial phase of translation initiation and the effect of GE81112 during the transition of a preIC to a 30SIC. (A) Overview on the interaction of the three IFs on the 30S subunit as observed in the cryo-EM structure of a 30S in complex with IF1 (green), IF2 (yellow), IF3 (magenta), P-tRNA (blue), and mRNA (60). (B) Zoom-in on the binding site of IF1 and IF3, near helices h44 (light green), h23 (pale blue), h24a (cyan), and h45 (orange) of 16S rRNA; the residues that acquire an alternative conformation in the presence of GE81112 are in red. (C) Scheme illustrating the molecular events suggested by the findings of the present study. (Left) A 30S preIC containing the three IFs and a not yet base-paired initiator tRNA and mRNA bears a flexible h44/h45/h24a interface. (Upper Center) If tRNA and mRNA are canonical, P-site decoding occurs to yield a bona fide 30SIC in which the h44/h45/h24a interface is engaged (locked). (Right) The affinity of this complex for IF3 is reduced, and the complex is amenable for docking by the 50S subunit and for undergoing 30SIC→70SIC transition with concomitant dissociation of the three IFs. (Lower Center) If the ligands in the 30S preIC are noncanonical or if GE81112 (or streptomycin) is present, a structurally faulty complex 30SIC* is formed in which proper codon–anticodon interaction does not occur. This complex contains a disengaged h44/h45/h24a structure, binds IF3 more tightly, and is unsuitable for docking with 50S subunits. Further details can be found in the text.