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The pre-translocation (PRE) complex of the ribosome can undergo spontaneous fluctuations of mRNA and tRNAs between classical and hybrid states, and occupation of the hybrid tRNA positions has been proposed to precede translocation. The classic and hybrid state tRNA positions been extensively characterized when the ribosome is stalled along the messenger RNA by either the absence or the delayed addition of elongation factor G (EF-G), or by the presence of antibiotics or GTP analogs that block translocation. Surprisingly, during multiple ongoing elongation cycles when both EF-G and ternary complexes are present, we found that tRNA positions in PRE complex ribosome do not fluctuate. Instead, they adopt a stationary intermediate structure between the stalled classical and hybrid tRNA positions, as indicated by single molecule fluorescence resonance energy transfer (FRET) between adjacent tRNAs and between A-site tRNA and ribosomal protein L11. These results indicate that EF-G promotes the formation of an intermediate structure during ongoing translation.

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2.2 Introduction

During protein synthesis, tRNAs successively occupy three sites on the ribosome: A (aminoacyl), P (peptidyl), and E (exit). After an aminoacylated tRNA (aa-tRNA) enters the A-site, a new peptide bond is formed as the nascent peptide chain is transferred from the P-site tRNA to the A-site tRNA. Translocation of the resulting pre-translocation (PRE) complex of the ribosome, which is a shift of the mRNA by one 3-base codon relative to the ribosome and movement of the tRNAs from the A and P sites to the P and E sites, respectively, is catalyzed by elongation factor G (EF- G). The resulting post-translocation (POST) complex awaits delivery of the next aa- tRNA into the A-site to start a subsequent cycle of elongation.

Although ribosomes in the PRE complex can undergo spontaneous translocation, they do so at a rate orders of magnitude slower than that achieved by EF-G catalysis (Cukras et al., 2003; Fredrick and Noller, 2003; Gavrilova and Spirin, 1971; Gavrilova et al., 1976; Pestka, 1969). The stalled PRE complex formed in the absence of EF-G fluctuates between so-called “classical” and “hybrid” states (Bretscher,1968; Moazed and Noller, 1989; Blanchard et al., 2004). In this earlier work, the “classical” tRNA position referred to the A/A, P/P conformation of bound tRNAs, where the first and second letters represent binding sites on the 30S and 50S subunits, respectively, and the “hybrid” tRNA position referred to a conformation containing tilted tRNAs, in which the tRNAs bound in the A/P and P/E state.

The hybrid state was proposed to be an intermediate between the classical PRE state and the POST complex in which the tRNAs are bound in the P/P and E/E positions. Subsequently, the definition of the hybrid state has broadened, largely as a result of structural studies (Brilot et al., 2013; Fischer et al., 2010; Ramrath et al., 2013) and FRET measurements both at the single molecule (Adio et al., 2015; Chen et

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al., 2011, 2013, Cornish et al., 2009a, 2009b, Fei et al., 2008, 2009, 2011, Munro et al., 2007b, 2010a) and ensemble (Belardinelli et al., 2016; Sharma and Chowdhury, 2011) levels. These studies have led to the identification of several distinct hybrid states of the PRE complex containing tRNAs in somewhat different positions on the ribosomes along the path between the classical PRE complex and the POST complex. The large and small subunits exhibit internal rotational movements, and the two subunits rotate ~8° relative to each other(Agirrezabala et al., 2008; Fischer et al., 2010). Frank and Gonzalez (2010) proposed that tRNA tilting and subunit rotations could be considered as coupled, defining two main global states: classical un-rotated states with non-tilted tRNAs, and hybrid rotated states with tilted tRNAs (Frank and Gonzalez, 2010). However, recent evidence demonstrates that tRNA tilting, subunit rotations, and L1 stalk motions are temporally distinct processes (Belardinelli et al., 2016; Munro et al., 2010a, 2010b; Sharma et al., 2016) that are not tightly coupled.

Here we present single molecule fluorescence resonance energy transfer (FRET) measurements focused on changes in tRNA positions on the ribosome during EF-G catalyzed translocation, as determined by changes in FRET efficiency between two ribosome-bound tRNAs, and between a tRNA and L11, a ribosomal protein located near the A-site. Earlier studies of tRNA movement during EF-G catalyzed translocation, obtained with ribosomes that had been stalled or slowed by either the absence of EF-G·GTP (Fischer et al., 2010; Kim et al., 2007; Munro et al., 2007a; Ning et al., 2014; Wang et al., 2011), or the replacement of EF-G·GTP by EF- G·GDPNP (Cornish et al., 2009b; Fei et al., 2008, 2009, 2011), or the addition of antibiotics (Adio et al., 2015; Ermolenko et al., 2007; Lin et al., 2015; Zhou et al., 2014), supported the notion that classical-hybrid transition in PRE complexes plays an important role in translocation. However, by artificially stalling translation, these

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systems imperfectly model the translocation process during multiple ongoing elongation cycles, when EF-G·GTP and ternary complexes, (aa-tRNA·EF-Tu·GTP) bind in quick succession. A study that did observe ongoing elongation (Chen et al., 2013) at low EF-G concentrations observed extremely slow fluctuations, but monitored subunit rotations rather than tRNA positions.

In contrast to the results obtained with stalled PRE complexes, our previous single molecule FRET study gave results indicating that tRNA positions in PRE complexes do not fluctuate during ongoing translation of a full-length protein (Rosenblum et al., 2013). However, that study used a time resolution similar to the measured rate of classical-hybrid tRNA fluctuations, which could have prevented their detection, and was only performed at a single EF-G·GTP concentration. In the present work, we overcome these limitations by detecting ongoing translation of a model polypeptide at higher time resolution, and at varying levels of EF-G·GTP. Our new results, presented below, show that at EF-G·GTP concentrations close to that in cells, tRNA positions in PRE complexes do not fluctuate but rather adopt a structure with a single dominant FRET efficiency that falls between the values for the stalled classical and hybrid tRNA positions, or close to the classical value. Fluctuations between classical and hybrid tRNA positions in PRE complexes only become apparent at EF-G·GTP levels well below measured Km values for the translocation activity of EF-G (Pan et al., 2007; Savelsbergh et al., 2003).