Antibiotics That Bind to the a Site of the Large Ribosomal Subunit Can Induce Mrna Translocation

Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation DMITRI N. ERMOLENKO,1,5 PETER V. CORNISH,2 TAEKJIP HA,3 and HARRY F. NOLLER4 1Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA 2Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA 3Department of Physics and Howard Hughes Medical Institute, University of Illinois, Urbana, Illinois 61801, USA 4Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA ABSTRACT In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre- translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding. Keywords: antibiotics; Brownian ratchet mechanism; mRNA translocation; ribosome INTRODUCTION kle et al. 2011). Blocking intersubunit rotation by formation During protein synthesis, mRNA and tRNAs are moved of a disulfide cross-link abolishes translocation (Horan and through the ribosome by the dynamic process of transloca- Noller 2007). Single-molecule Förster Resonance Energy tion. Sequential movement of tRNAs from the A (aminoacyl) Transfer (smFRET) studies demonstrate that pre-transloca- site to the P (peptidyl) site to the E (exit) site is coupled to tion ribosomes undergo spontaneous intersubunit rotational movement of their associated codons in the mRNA (Spirin movement in the absence of ribosomal factors and GTP (Cornish et al. 2008), fluctuating between two conformations 2004). Translocation is catalyzed by a universally conserved corresponding to the classical and hybrid states of the tRNA elongation factor (EF-G in prokaryotes and EF-2 in eukary- translocational cycle (Fig. 1A; Blanchard et al. 2004; Ermo- otes) (Spirin 2004). lenko et al. 2007b; Munro et al. 2007; Fei et al. 2008). Howev- Ribosomal translocation takes place in two steps, in which er, under most experimental conditions, in the absence of EF- the acceptor ends of the tRNAs first move relative to the large G, these fluctuations do not result in productive translocation ribosomal subunit (50S in bacteria), from the classical A/A of mRNA and tRNAs on the small ribosomal subunit. EF-G and P/P states into hybrid A/P and P/E states, followed by transiently stabilizes the hybrid state conformation (Pan the movement of their anticodon ends on the small ribosomal et al. 2007; Spiegel et al. 2007; Ermolenko and Noller 2011) subunit (30S), coupled with mRNA movement, into the post- and catalyzes mRNA translocation during the reverse (clock- translocational P/P and E/E states (Fig. 1A; Moazed and wise) rotation of the small subunit (Ermolenko and Noller Noller 1989). While hybrid state formation can occur sponta- 2011). mRNA translocation is likely accompanied by addi- neously, translocation on the small subunit requires EF-G and tional conformational changes within the small ribosomal GTP (Moazed and Noller 1989). Formation of the hybrid state subunit, i.e., “swiveling” motion of the “head” relative to intermediate is accompanied by a counterclockwise rotation the “body” and the “platform” of the 30S, which is orthogo- of the small ribosomal subunit (Valle et al. 2003; Ermolenko nal to the intersubunit rotation (Schuwirth et al. 2005; Ratje et al. 2007a; Agirrezabala et al. 2008; Julian et al. 2008; Dun- et al. 2010; Dunkle et al. 2011; Zhou et al. 2012). Despite re- cent progress in studies of the translocation mechanism, the 5 Corresponding author question remains: How does EF-G convert unproductive E-mail [email protected] Article published online ahead of print. Article and publication date are at spontaneous fluctuations of the ribosome and tRNAs into http://www.rnajournal.org/cgi/doi/10.1261/rna.035964.112. efficient translocation of tRNA and mRNA? RNA (2013), 19:1–9. Published by Cold Spring Harbor Laboratory Press. Copyright © 2013 RNA Society. 1 Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Ermolenko et al. Here, we exploit the use of antibiotics as an example of rectification of ribosomal dynamics via ligand binding. Previously, an intriguing translocation phenomenon was discovered: The antibiotic sparsomycin, a peptidyl transferase inhibitor that binds to the large ribosomal subunit, was found to induce efficient translocation of tRNA and mRNA on the small subunit (Fredrick and Noller 2003). Early studies on the inhibitory action of sparsomycin showed that it in- creases the affinity of tRNA for the P site while interfering with binding to the A site (Monro et al. 1969; Pestka 1969; Lazaro et al. 1991). Consistent with these findings, crystallographic studies of sparsomycin bound to the large ribosomal subunit showed that its uracil moiety in- teracts with the 3′-terminal cytosine and adenine of the P-site tRNA, while its sul- fur-rich tail overlaps with the binding position of the aminoacyl moiety and terminal adenine of the A-site tRNA (Hansen et al. 2003). It was originally FIGURE 1. Antibiotic-induced translocation of mRNA. (A) Schematic depiction of intersubunit rotation and tRNA movement in pre-translocation ribosomes. (Movement of mRNA is not hypothesized that sparsomycin induces shown.) Under most experimental conditions, spontaneous, nonproductive forward and reverse translation by stabilization of the post- intersubunit rotation, coupled to fluctuations between the classical and hybrid states of tRNA translocation state of the ribosome via in- binding, does not result in mRNA translocation in the absence of EF-G. (B) Schematic illustration of hypothesized mechanism of sparsomycin-induced translocation (see details in the text). teraction with peptidyl-tRNA in the 50S Binding of sparsomycin to the 50S A site is indicated by a star. (C) Toeprinting assay of antibi- P site (Fredrick and Noller 2003). How- otic-induced mRNA translocation. A pre-translocation complex was made by binding deacylated ever, the observation that spontaneous Tyr Phe tRNA to the P site (lanes 1 and 10) and the peptidyl-tRNA analog N-Ac-Phe-tRNA to the A movement of the acceptor ends of tRNAs site (lane 2) in the presence of mRNA 301. Pre-translocation ribosomes were incubated with sparsomycin (lane 3), lincomycin (lane 4), clindamycin (lane 5), chloramphenicol (lane 6), blasticidin on the large subunit does not result in S (lane 7), and erythromycin (lane 8). A toeprint band at +16 corresponds to the pre-translocation efficient translocation on the small sub- complex, a band at +19 is the product of translocation, and a band at +14 is the product of direct unit (Fig. 1A; Moazed and Noller 1989; Phe binding of N-Ac-Phe-tRNA to the upstream Phe codon (lane 9). Blanchard et al. 2004; Fei et al. 2008) sug- gests that this explanation may be in- The spontaneity of intersubunit rotational movement sufficient. An alternative possibility is that sparsomycin prompted several groups to apply the theoretical framework induces ribosomal translocation by sterically blocking the re- of the Brownian ratchet mechanism to the analysis of ribo- verse movement of peptidyl-tRNA from the hybrid A/P to the somal translocation (Frank and Gonzalez 2010; Ratje et al. classical A/A state as the ribosome spontaneously fluctuates 2010; Dunkle et al. 2011). In Brownian ratchet models, between the rotated and nonrotated conformations (Fig. unidirectional movement of macromolecules is enabled by li- 1B). In this model, sparsomycin plays the role of a pawl in gand binding and chemical reactions that rectify spontaneous a Brownian ratchet. To test this possibility, we examined thermal fluctuations of macromolecules by preferentially the ability of other antibiotics that bind to the 50S A site locking in a preferred state (Howard 2006). (By “rectify,” but are chemically different from sparsomycin, to induce we mean conversion of movement, which periodically revers- mRNA translocation. es direction, to unidirectional motion.) Thus, ligand binding is analogous to the pawl of a mechanical ratchet. However, RESULTS the hypothesis that the ribosome is a Brownian ratchet machine whose spontaneous dynamics can be converted into Antibiotics that bind to the 50S A site induce translation (Spirin 2004; Frank and Gonzalez 2010; Ratje mRNA translocation et al. 2010; Dunkle et al. 2011) has not been rigorously tested. Moreover, the exact nature of the pawl of the translocation Biochemical and crystallographic studies have identified

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