Blasticidin S inhibits by trapping deformed tRNA on the

Egor Svidritskiya, Clarence Lingb, Dmitri N. Ermolenkob,1, and Andrei A. Korosteleva,1

aRNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605; and bDepartment of Biochemistry and Biophysics, Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642

Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved June 3, 2013 (received for review March 14, 2013)

The blasticidin S (BlaS) is a potent inhibitor of protein to BlaS were shown to be also resistant to sparsomycin and pu- synthesis in bacteria and eukaryotes. We have determined a 3.4-Å romycin, further suggesting that the inhibitory mode of action of crystal structure of BlaS bound to a 70S·tRNA ribosome complex BlaS may involve the binding of the drug to the A site (12). Thus, and performed biochemical and single-molecule FRET experiments the mechanism of BlaS action remains a conundrum: how can to determine the mechanism of action of the antibiotic. We find BlaS, whose binding site overlaps with that for P-site tRNA, that BlaS enhances tRNA binding to the P site of the large ribo- stabilize the binding of the pentanucleotide mimic of the 3′ end somal subunit and slows down spontaneous intersubunit rotation of tRNA to the P site on the 50S subunit (8) and inhibit binding in pretranslocation . However, the antibiotic has negli- of tRNA analogs and to the A site? gible effect on elongation factor G catalyzed translocation of tRNA In this work, we elucidate the mechanism of BlaS action by and mRNA. The crystal structure of the antibiotic–ribosome com- X-ray crystallography, single-molecule Förster resonance energy plex reveals that BlaS impedes protein synthesis through a unique transfer (smFRET) and biochemical assays. We show that BlaS mechanism by bending the 3′ terminus of the P-site tRNA toward inhibits protein synthesis through a unique mechanism by bend- the A site of the large ribosomal subunit. Biochemical experiments ing the CCA end of P-site tRNA toward the A site of the large demonstrate that stabilization of the deformed conformation of subunit. Stabilization of this unusual, deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis P-site tRNA by BlaS results in inhibition of peptide bond for- – by release factors and, to a lesser extent, peptide bond formation. mation and peptidyl tRNA hydrolysis by release factors. BIOCHEMISTRY Results and Discussion peptidyl transfer | ribosome crystal structure | termination inhibitor | translation inhibitor | translation termination BlaS Stabilizes tRNA in the P Site of the Large Ribosomal Subunit. Early biochemical experiments showed that BlaS stabilizes binding of tRNA analogs to the 50S P site (8), which appears to conflict he growing problem of pathogen resistance to existing anti- with crystallographic data showing that the binding sites of BlaS Tbacterials prompts a search for alternative modes of inhibiting and the CCA end of P-site tRNA overlap (5). To reexamine the bacterial growth. The development of new drugs can be facilitated effect of BlaS on P-site binding, we used a filter-binding assay to by understanding the mechanisms of action of known antibiotics monitor the binding of N-formyl-methionyl-tRNAfMet (fMet- (1). Because of its central role in cell metabolism, the ribosome is tRNAfMet)tothePsiteofE. coli 70S ribosomes preincubated with the target of numerous inhibitors that bind to various sites on the fi ribosome and interfere with different steps of protein synthesis. various concentrations of BlaS in the presence of de ned mRNA. Importantly, in contrast to previous biochemical experiments (8, One of the predominant modes of action of ribosomal antibiotics 35 fMet is the inhibition of peptide bond formation. A majority of peptidyl- 9), we used full-length initiator [ S]-fMet-tRNA instead of short tRNA analogs, such as CACCA. No inhibition of tRNA transferase inhibitors, including the widely used antibacterials ≤ chloramphenicol, linezolid, and the lincosamides lincomycin and binding was observed at concentrations of the antibiotic 1mM (Fig. 1B). BlaS interfered with [35S]-fMet-tRNAfMet binding to the clindamycin, bind to the A site of the large ribosomal subunit at ∼ the peptidyl-transferase center of the ribosome (2–4). By contrast, ribosome only at millimolar antibiotic concentrations (IC50 20 blasticidin S (BlaS) binds to the P site of the large subunit (5) and mM), i.e., concentrations that exceed the inhibition constant of – fi inhibits the peptidyl-transferase reaction through a distinct mech- BlaS for peptidyl transfer (200 400 nM; see 13, 14) by nearly ve anism, which is still poorly understood. orders of magnitude. Notably, these concentrations exceed by up BlaS, produced by some Streptomyces species, is a nucleoside to four orders of magnitude the concentration of BlaS sufficient to analog consisting of a bonded to a pyranose ring and abolish growth of bacterial and eukaryotic cells (7, 15–17). The attached to an N-methyl-guanidine tail (Fig. 1A). BlaS has long destabilizing effect of large amounts of the antibiotic on tRNA been known to be a potent inhibitor of protein synthesis in binding is likely due to BlaS binding to a second, low-affinity site bacteria and eukaryotes (6, 7). A crystal structure of the 50S on the ribosome observed in the structure of the large ribosomal subunit from Haloarcula marismortui bound to BlaS in the ab- subunit from H. marismortui crystallized in the presence of 1–10 sence of tRNA revealed that BlaS occupies the P site of the large mM BlaS (5). In sum, the antibiotic does not hinder tRNA binding subunit (5). Two molecules of BlaS interact with the P loop and to the P site of the ribosome at concentrations that are inhibitory form base pairs with the universally conserved G2251 and G2252 for protein synthesis and cell growth. of 23S ribosomal RNA (Escherichia coli 70S ribosome number- ing is used throughout this article). These base pairing inter- actions closely mimic those of the two cytosine residues of the Author contributions: D.N.E. and A.A.K. designed research; E.S. and C.L. performed re- conserved CCA 3′ terminus of tRNA bound in the P site. Based search; E.S., C.L., D.N.E., and A.A.K. analyzed data; and E.S., C.L., D.N.E., and A.A.K. wrote on the finding that two molecules of BlaS mimic the cytosine res- the paper. idues of the CCA 3′ end of the P-site tRNA, BlaS was proposed The authors declare no conflict of interest. to inhibit protein synthesis by competing with tRNA binding This article is a PNAS Direct Submission. to the P site (5). However, early biochemical studies demon- Data deposition: The atomic coordinates and structure factors have been deposited in the strated that BlaS enhances P-site binding of aminoacyl-CACCA, Protein Data Bank, www.pdb.org (PDB ID codes 4L6J, 4L6K, 4L6L, and 4L6M). ′ which mimics the 3 end of aminoacylated tRNA (8) and modestly 1To whom correspondence may be addressed. E-mail: [email protected] inhibits the binding of aminoacyl-CACCA to the A site (8, 9). or [email protected]. Moreover, BlaS competes with A-site binding antibiotics, such as This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. sparsomycin (10) and (11). Mammalian cells resistant 1073/pnas.1304922110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1304922110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 (23). Using FRET in combination with total internal reflection microscopy, we have reported spontaneous fluctuations in single pretranslocation ribosomes between ∼0.6 and ∼0.4 FRET values, which correspond to the classical (nonrotated) and hybrid (ro- tated) states, respectively (22). We examined how BlaS affects the affinity of tRNA to the 50S P site by monitoring the dynamics of single-tRNA fluctuations between the classical P/P and hybrid P/E states. To this end, we added tRNAPhe to the P site of S6-Cy5/L9-Cy3-labeled ribosomes in the presence of defined mRNA and then flowed 1 mM BlaS into a sample cell. In the absence of BlaS, ribosomes underwent frequent intersubunit rotation and spent ∼70% of the time in the rotated, hybrid-state conformation (Fig. 2B), consistent with prior observations (18–20). Addition of BlaS dramatically increased the fraction of nonrotated ribosomes, from ∼30% to ∼80% (Fig. 2C and Table S1). The rate of clockwise rotation of the 30S subunit Fig. 1. Effect of BlaS on fMet-tRNAfMet binding to the P site of the 70S ri- coupled to tRNA transition from the hybrid P/E to the classical bosome. (A) Chemical structure of BlaS. (B) Binding of [35S]-fMet-tRNAfMet to P/P state was unaffected by BlaS. By contrast, BlaS addition the ribosome in the presence of mRNA and various concentrations of BlaS resulted in a fivefold reduction of the rate of counterclockwise measured by a filter-binding assay. Bars show the P-site occupancy by [35S]- rotation coupled with transition of tRNA from the classical P/P to fMet-tRNAfMet relative to that in the ribosomes not treated with the anti- the hybrid P/E state (Table S1). Ribosomes containing a single biotic (the occupancy in the absence of the antibiotic is set to 1.0). Error bars elongator tRNAMet or initiator tRNAfMet instead of tRNAPhe show SDs calculated from triplicate measurements. behaved similarly (Fig. S1), underscoring that the effect of BlaS does not depend on tRNA identity. Inhibition of counterclock- fi wise intersubunit rotation was recently observed in the presence To further explore the effects of BlaS on tRNA af nity to the of another peptidyl-transferase inhibitor, sparsomycin, which is 50S P site, we asked whether BlaS affects the dynamics of ribo- also known to enhance tRNA binding to the 50S P site (25). We some-bound tRNAs, whose translocation through the ribosome is conclude that BlaS inhibits the transition of deacylated tRNA essential for protein synthesis. tRNA translocation, which is cat- into the hybrid P/E state (Fig. 2C) via stabilization of the acceptor alyzed by elongation factor G [EF-G (EF-2 in eukaryotes)], occurs end of tRNA in the 50S P site, consistent with previous bio- fi in two steps. The acceptor ends of tRNAs rst move relative to chemical data (8, 9). the large ribosomal subunit, from the classical A/A and P/P states into hybrid A/P and P/E states (Fig. 2A). The next step is the Effects of BlaS on the Dynamics of Pretranslocation Ribosomes and movement of anticodon stem loops of tRNAs on the small ribo- mRNA Translocation. We next tested the effect of BlaS on spon- somal subunit into posttranslocation P/P and E/E states. Whereas taneous intersubunit dynamics of authentic pretranslocation tRNA translocation on the small subunit requires EF-G and GTP, ribosomes containing tRNAs in both A and P sites (Fig. 2A). the movement of acceptor stems of peptidyl- and deacylated Similarly to ribosomes containing a single tRNA in the P site, the tRNAs on the large subunit occurs spontaneously, fluctuating majority (∼70%) of pretranslocation ribosomes containing the between the classical (A/A and P/P) and hybrid (A/P and P/E) peptidyl-tRNA analog (18, 19, 26) N-acetyl-Phe-tRNAPhe in the states (18–20). These spontaneous fluctuations of tRNAs are A site and deacylated elongator tRNAMet in the P site adopt coupled to a rotation of the small ribosomal subunit relative to the the rotated, hybrid-state conformation (Fig. 3A). In the presence large subunit between the nonrotated and rotated conformations, of 1 mM BlaS, the majority of pretranslocation ribosomes respectively (21–24). We previously developed a method to follow (∼60%) remained in the rotated state. This was surprising be- the intersubunit dynamics of the ribosome by measuring smFRET cause BlaS dramatically shifted the distribution of ribosomes between fluorophores attached to protein S6 on the platform of containing a single deacylated tRNAMet toward the nonrotated, the small ribosomal subunit and protein L9 on the large subunit classical-state conformation (Fig. S1). However, kinetic analysis

Fig. 2. Blasticidin S inhibits counterclockwise rota- tion of the 30S subunit coupled to the movement of deacylated tRNA into the hybrid P/E state. (A) Schematic depiction of intersubunit rotation, which is measured by smFRET, and tRNA movement dur- ing ribosomal translocation. (B and C) Histograms showing distributions of FRET values in ribosomes containing deacylated tRNAPhe in the P site in the absence (B) or presence (C) of BlaS. N, number of single-molecule FRET traces compiled.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1304922110 Svidritskiy et al. Downloaded by guest on September 26, 2021 of single-molecule traces revealed that BlaS reduced the rates The Crystal Structure of the 70S·tRNA Complex Bound with BlaS of both clockwise and counterclockwise rotation approximately Reveals That BlaS Induces a Deformed Conformation of P-site tRNA. sixfold, leaving the equilibrium constant (the ratio of rates for A previous crystal structure of BlaS bound to the vacant 50S counterclockwise and clockwise intersubunit rotation) largely subunit of the H. marismortui ribosome revealed that the binding unaffected (Fig. S2 and Table S1). Inhibition of counterclock- site for two molecules of BlaS overlaps with the binding site for P- wise rotation of the 30S subunit coupled to transition of tRNA tRNA (5). How can the enhancement of tRNA binding to the 50S into hybrid states is likely due to stabilization of the acceptor end P site observed in our smFRET experiments be achieved by an of deacylated tRNA in the 50S P site by BlaS, which fixes P-site antibiotic that appears to compete with tRNA? To resolve this tRNA in the P/P classical state (Fig. 2A). This echoes our obser- paradox, we determined a 3.4 Å crystal structure of BlaS bound vation for ribosomes containing a single deacylated tRNA in the to the 70S Thermus thermophilus ribosome in the presence of P site (Fig. 2B). By contrast, inhibition of clockwise rotation of deacylated tRNAfMet molecules in the P and E sites (Fig. 4A and the 30S evidently occurs only in the presence of an A-site tRNA Table S3). In previously reported structures of 70S elongation (31, and is likely due to stabilization of the acceptor end of peptidyl- 32) or termination (33–35) complexes, the overall conformations tRNA in the 50S P site by BlaS, which fixes A-site tRNA in the of deacylated initiator tRNAfMet are nearly identical to those of hybrid A/P state (Fig. 2A). Thus, in pretranslocation ribosomes elongator tRNAs and peptidyl-tRNA analogs. Together with the containing tRNAs in both A and P sites, BlaS inhibits both the finding that the action of BlaS is unlikely to depend on the identity forward and reverse transitions between the classical and hybrid of tRNA (Fig. S1), this suggests that our crystal structure reflects states of tRNA binding. the structural mechanism of action of BlaS on initiating and Because BlaS significantly slows down tRNA and intersubunit elongating ribosomes. dynamics in pretranslocation ribosomes in the absence of EF-G, Unlike in the crystal structure of the vacant 50S subunit bound we next asked whether BlaS also inhibits EF-G-catalyzed trans- with two molecules of BlaS, only one molecule is found in the location of mRNA and tRNA. EF-G·GTP transiently stabilizes 70S·tRNA complex (Fig. 4B). The conformation of BlaS is the rotated, hybrid-state conformation (27) and induces trans- similar to that of molecule 1 in the 50S crystal structure (5); the location of the anticodon stem loops of tRNA and cognate codons all-atom rms difference between them is 0.57 Å, similar to the of mRNA from A and P to P and E sites, respectively (26). Here, coordinate error at 3.4 Å resolution. The N-methyl-guanidine we followed the kinetics of mRNA translocation in the presence of moiety of BlaS is stacked on the base of A2439 of 23S ribosomal EF-G·GTP and BlaS by measuring the fluorescence quenching of RNA and is stabilized by interaction with the negatively charged a fluorescein dye attached to the 3′ end of the mRNA as it moves pocket formed by the phosphate groups of A2439, A2600, and

within the ribosome (28). Pretranslocation ribosomes assembled C2601 (Fig. 4C). A single binding site for BlaS next to G2251 BIOCHEMISTRY with deacylated tRNAMet in the P site, N-acetyl-Phe-tRNAPhe in agrees with chemical probing and mutational data. In particular, the A site, and fluorescein-labeled mRNA were mixed with EF- interaction of the N-methyl-guanidine tail of BlaS with A2439 G·GTP in a stopped-flow apparatus. mRNA translocation was explains protection of A2439 from chemical modification in the − detected by rapid (3.7 ± 0.6 s 1) quenching of the fluorescein dye presence of BlaS (36, 37). Mutation of U2438 of 23S ribosomal (Fig. 3C and Table S2). When pretranslocation ribosomes were RNA confers BlaS resistance in the archaeon Halobacterium preincubated with BlaS, the apparent rate of translocation was halobium (38). This can be explained by disruption of the U2438: − only slightly reduced (2.6 ± 0.4 s 1)(Fig.3C and Table S2). A2071 base pair, which likely results in displacement of the ad- Therefore, despite significant inhibition of spontaneous ribosome jacent A2439 and disruption of the interaction between A2439 and tRNA dynamics by BlaS, EF-G-catalyzed translocation is al- and BlaS. most unaffected by the presence of the antibiotic. Notably, per- In the 70S structure, BlaS forms a base pair with G2251 of the turbations of the P site of the large subunit by mutations in the P 23S ribosomal RNA and intercalates between C74 and A76 of the loop of 23S ribosomal RNA (G2252C and G2251C) were also CCA end of P-site tRNA. This interaction replaces the base pair- shown to considerably affect the frequency of spontaneous tRNA ing between G2251 and C75 observed in antibiotic-free 70S·tRNA fluctuations between classical and hybrid states (29) without dra- complexes (31, 32, 39), resulting in displacement of the acceptor matic effects on the rate of EF-G-induced translocation (30). arm and the CCA end of the P-tRNA toward the A site (Fig. 4D). Together, these observations suggest that formation of the hybrid The ribose-phosphate backbone of C75 is shifted by more than 7 Å, state intermediate is accelerated by EF-G and is not a rate-limiting whereas those of the adjacent C74 and A76 are displaced by ∼4 step of translocation. and ∼2 Å, respectively. This large local rearrangement results in

Fig. 3. Effects of BlaS on spontaneous intersubunit rotation in pretranslocation ribosomes and kinetics of EF-G-catalyzed mRNA translocation. (A and B) smFRET distribution histograms for pretranslocation ribosomes containing tRNAMet and N-acetyl-Phe-tRNAPhe in the P and A sites, respectively, in the absence (A) or presence (B) of BlaS. (C) Kinetics of EF-G-catalyzed translocation of mRNA in ribosomes preincubated with BlaS (red) or not treated with the antibiotic (blue). Kinetic measurements were performed in presteady-state stopped-flow kinetic experiments by the quenching of fluorescein attached to the mRNA. Pretranslocation ribosomes containing deacylated elongator tRNAMet in the P site and N-acetyl-Phe-tRNAPhe in the A site were mixed with GTP and EF-G. Double-exponential fits for fluorescein quenching are black curves.

Svidritskiy et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 Fig. 4. Crystal structure of T. thermophilus 70S ri- bosome bound with BlaS in the presence of tRNAfMet inthePandEsites.(A) Positions of BlaS (blue), P-site (orange), and E-site (red) tRNAs; 30S subunit is in cyan and 50S subunit in gray. (B)Un-

biased Fobs-Fcalc density corresponding to BlaS and the CCA tail of the P-site tRNA. (C)Interactionsof BlaS with the P site of the 50S subunit. (D)Super- position of tRNA in the 70S·tRNA·BlaS complex (orange, this work) with that from the antibiotic- free 70S·tRNA complex (green, ref. 31), demon- strating the BlaS-induced shift of the 3′ end toward the A site.

a ∼4o rotation of the acceptor arm of the tRNA toward the 50S A mechanistically similar to peptidyl-tRNA hydrolysis mediated site. The orientation of the rest of the P-site tRNA is nearly by release factors 1 and 2 (RF1 and RF2), which bind to the identical to that of the tRNA bound in the absence of BlaS (Fig. ribosomal A site in response to stop codons and catalyze pep- 4D). The movement of the CCA backbone caused by BlaS suggests tidyl-tRNA hydrolysis (40–43). Release factors are directly in- that BlaS competition with A-site binding antibiotics (10, 11) is volved in peptidyl-tRNA hydrolysis by providing a catalytic induced allosterically. group located in the universally conserved GGQ motif (40–43). Whereas C74 is rearranged with respect to the 50S P site, it We reasoned that in addition to its peptidyl-transfer inhibitory remains paired with G2252, whose base is moved by more than activity, BlaS may be a translation termination inhibitor. Super- 1 Å codirectionally with the base of C74 (Fig. 4B). The ribose- imposition of the BlaS-bound 70S structure with structures of phosphate backbone of the P loop is not affected by BlaS. The 70S termination complexes (34, 35, 44) revealed that the dis- base of the terminal nucleotide A76, which is stacked on BlaS, torted CCA end of the P-site tRNA in the BlaS-bound ribosomal remains in a position similar to that in the absence of BlaS complex substantially overlaps with the binding site for the (31, 32, 39). Intercalation of the cytosine moiety of BlaS be- GGQ-containing region of domain 3 of the release factors (Fig. tween the bases of C74 and A76 likely compensates for the loss 5A). This suggests that distortion of the P-site tRNA by BlaS may of stacking interaction of these nucleotides with C75. In addi- interfere with binding of release factors to the ribosome and/or tion, the 2′-hydroxyl group of C75 is positioned to hydrogen prevent proper positioning of the GGQ motif for peptidyl- bond with the N4 position of the cytosine ring of the antibiotic, tRNA hydrolysis. providing additional stabilization to the distorted conformation To test our hypothesis, we monitored the effect of BlaS on of tRNA. In sum, the stabilizing interactions that BlaS forms RF1-mediated peptide release from 70S ribosomes. We found with ribosomal RNA and P-site tRNA explain the observed that BlaS dramatically inhibits RF1-mediated [35S]-fMet release stabilization of the acceptor arm in the P site (Fig. 2B). from [35S]-fMet-tRNAfMet bound to the P site of the ribosome in Our crystal structure provides insights into the mechanism of the presence of mRNA containing a UAA stop codon adjacent inhibition of peptidyl transfer by BlaS. Although the adenine moi- to an AUG codon. Specifically, BlaS nearly abolishes RF1- ety of A76 of the P-site tRNA is positioned to form an A-minor mediated release (Fig. 6A) at concentrations that were only interaction with the A2450–C2063 bp of 23S ribosomal RNA (as modestly inhibitory to peptidyl transfer of [35S]-fMet to puro- in antibiotic-free complexes; see refs. 31, 32, 39), rearrangement mycin (Fig. 6B), which is an A-site antibiotic that mimics the 3′ of the CCA backbone results in detachment of the ribose of A76 end of an aminoacyl-tRNA. The apparent inhibition constant from C2063. Rearrangement of the ribose moiety of A76 may (Ki) of 182 ± 39 nM for BlaS-dependent inhibition of the pu- result in suboptimal positioning of the peptidyl-tRNA and ami- romycin reaction determined from our measurements is consis- noacyl-tRNA substrates for nucleophilic attack, leading to in- tent with previous estimates of a Ki between 200 nM (13) and 380 hibition of peptidyl transfer by BlaS. nM (14). Remarkably, the apparent Ki for the RF1-mediated release (32 ± 18 nM) is almost one order of magnitude lower BlaS Strongly Inhibits Peptide Release Mediated by RF1. The nu- than the Ki for the puromycin reaction. It is noteworthy that cleophilic substitution reaction of peptide-bond formation is the sensitivity of the termination assay does not permit

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1304922110 Svidritskiy et al. Downloaded by guest on September 26, 2021 Our smFRET measurements show that BlaS slows down the intersubunit rotation in pretranslocation ribosomes that is coupled to spontaneous movement of tRNAs on the large subunit (Fig. 3A). This observation is consistent with early biochemical data demonstrating that BlaS increases tRNA affinity toward the 50S P site (8). Despite the inhibitory effect on tRNA fluctuations in a pretranslocation complex, EF-G-catalyzed translocation is af- fected only modestly (Fig. 3B). Our data therefore rule out the possibility that inhibition of translocation is a mode of BlaS action in vivo. Instead, our biochemical experiments suggest that BlaS may specifically target the termination step of protein synthesis. Fig. 5. Superposition of the 70S·BlaS structure (this work) with structures of We found that BlaS efficiently inhibits peptide release medi- 70S complexes whose A site is occupied by RF1 (35) or aminoacyl-tRNA an- ated by release factor RF1 during termination of protein synthesis alog (32). The superposition demonstrates that steric hindrance in the and, to a lesser extent, peptidyl transfer during translation elon- presence of BlaS is more pronounced in the case of release factor (A, green) gation. Inhibition of these steps of translation likely originates than in the case of aminoacyl-tRNA (B, magenta). from a combination of two effects. First, the shift of the distorted CCA end of the P-site tRNA may occlude access of aminoacyl- tRNA and release factors into the A site on the 50S subunit (Figs. measurements of peptide release at concentrations of the pre- 4 and 5). The stronger inhibition of RF1-mediated peptide re- termination complex below 30 nM. Thus, the actual Ki for BlaS K lease by BlaS may result from a more pronounced steric in- inhibition of termination may be even lower than the apparent i terference of the distorted CCA end with the GGQ-containing (∼32 nM) determined from our experiments. Hence, our bio- fi region of release factors than with the A-site aminoacyl-tRNA chemical experiments demonstrate that BlaS is a more ef cient (Fig. 5). Second, the terminal adenosine residue of the P-site inhibitor of translation termination than of peptidyl transfer C D tRNA is shifted from the position it occupies in the absence of (Fig. 6 and ). the antibiotic (Fig. 4D). This rearrangement likely results in poor positioning of the peptidyl-tRNA substrate for nucleophilic attack Conclusions by aminoacyl-tRNA during peptidyl transfer or by a water mol- fi Our ndings demonstrate that BlaS inhibits translation via a ecule during translation termination.

unique molecular mechanism that has not been previously docu- The strong inhibition of the translation termination step by BIOCHEMISTRY mented for any antibiotic. The crystal structure of the BlaS-bound BlaS is noteworthy because no specific inhibitors of translation 70S ribosome complex reveals that unlike most peptidyl-transfer termination have been identified so far (2, 45–47). Some inhib- inhibitors, which bind in the vicinity of the A site of the 50S itors of peptide bond formation have been shown to also inhibit subunit, BlaS binds to the P site of the 50S subunit and bends the peptide release (2), but none has been quantitatively demon- CCA end of the P-site tRNA toward the A site. The dramatic strated to inhibit the latter step to a greater degree than peptidyl (>7 Å) shift of the ribose-phosphate backbone of C75 of the P-site transfer (48). A large number of inherited and acquired diseases tRNA underscores the conformational flexibility of tRNA. This are attributed to the occurrence of a premature termination observation expands our knowledge about the conformational codon within the normal open reading frame, which precludes space that tRNAs can sample during translation. More impor- synthesis of full-length protein (49, 50). Because BlaS pre- tantly, the distortion of the P-site tRNA explains the puzzling, dominantly targets peptide release and also inhibits protein previously reported ability of BlaS to interfere with binding of synthesis in eukaryotes (15, 17), our findings may potentially ribosomal ligands to the A site (8–11). serve as a platform for the development of therapeutics that

Fig. 6. BlaS is a potent inhibitor of RF1-mediated peptide release. (A and B) Time progress curves for RF1-mediated release (A) and peptidyl transfer to puromycin (B), respectively, in the absence (blue) or presence (red) of 800 nM BlaS. Pretermination complex was formed by incubating ribosomes with [35S]-fMet-tRNAfMet and mRNA containing the UAA termination codon next to the AUG codon. Then, pretermination ribosomes were incubated with ei- ther RF1 (A) or puromycin (B) and amounts of [35S]- labeled release product were determined at several time points. Error bars represent SDs obtained from duplicate experiments. (C and D) Dependence of

catalytic rates (kobs) for RF1-mediated release (C) and puromycin reaction (D) on BlaS concentration.

Ki for the peptide release and puromycin reaction was determined by hyperbola fitting (red) of kobs values obtained at different concentrations of BlaS. Error bars represent residuals of single-exponential

fitting of kobs values to time progress curves.

Svidritskiy et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 specifically suppress translation termination in human diseases and 23ID-D (Advanced Photon Source at Argonne National Laboratory) and associated with premature termination codons. X25 (National Synchrotron Light Source at Brookhaven National Laboratory, US Department of Energy, under Contract No. DE-AC02-98CH10886) for Materials and Methods assistance with X-ray data collection; Jillian Dann and Enea Salsi for fi Δ Δ Materials and methods are described in detail in SI Materials and Methods. assistance with puri cation of S6 and L9 ribosomes and smFRET experi- Ribosomes, aminoacylated tRNAs, EF-G, and RF1 were prepared as previously ments, respectively; Peter Cornish for sharing MatLab scripts and advice on described (23, 34, 44). Single-molecule FRET measurements were taken using total internal reflection microscopy; Alexei V. Korennykh for stimulating a prism-type total internal reflection (TIR) microscope (22). Structure de- discussions; and Harry F. Noller for comments on the manuscript. The study termination was performed using molecular replacement and yielded a was supported by US National Institute of Health Grant GM-099719 (to D. structure with R/Rfree of 0.232/0.268. N.E.) and Grant P30 GM092424 (to the Center for RNA Biology at University of Rochester). A.A.K. was supported by the Worcester Foundation for Biomedical ACKNOWLEDGMENTS. We thank Rohini Madireddy for assistance with Research and University of Massachusetts Medical School Center for purification of RF1 and 70S ribosomes; staff members of beam lines 23ID-B AIDS Research.

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