Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state

Abstract

After four decades of research aimed at understanding tRNA selection on the ribosome, the mechanism by which ribosomal ambiguity (ram) mutations promote miscoding remains unclear. Here, we present two X-ray crystal structures of the Thermus thermophilus 70S ribosome containing 16S rRNA ram mutations, G347U and G299A. Each of these mutations causes miscoding in vivo and stimulates elongation factor thermo unstable (EF-Tu)-dependent GTP hydrolysis in vitro. Mutation G299A is located near the interface of ribosomal proteins S4 and S5 on the solvent side of the subunit, whereas G347U is located 77 Å distant, at intersubunit bridge B8, close to where EF-Tu engages the ribosome. Despite these disparate locations, both mutations induce almost identical structural rearrangements that disrupt the B8 bridge-namely, the interaction of h8/h14 with L14 and L19. This conformation most closely resembles that seen upon EF-Tu·GTP·aminoacyl-tRNA binding to the 70S ribosome. These data provide evidence that disruption and/or distortion of B8 is an important aspect of GTPase activation. We propose that, by destabilizing B8, G299A and G347U reduce the energetic cost of attaining the GTPase-activated state and thereby decrease the stringency of decoding. This previously unappreciated role for B8 in controlling the decoding process may hold relevance for many other ribosomal mutations known to influence translational fidelity.T he molecular mechanisms controlling the fidelity of DNA replication, transcription, and translation have been areas of intense interest since the discovery of the genetic code. Thermodynamic differences between standard Watson-Crick and alternative (e.g., wobble) base pairs in solution are insufficient to explain the high fidelity for any of the three polymerase reactions of the central dogma (1), indicating an active role for the enzymes in substrate selectivity (1-3). Mechanistic studies of polymerases have revealed some common themes, such as the specific recognition of Watson-Crick base pair geometry, larger forward rate constants for correct substrates (induced fit), separate opportunities for incorrect substrate rejection (kinetic proofreading), and postincorporation correction mechanisms (1-5).During translation, the ribosome must select aminoacyl-tRNA (aa-tRNA) substrates based on the mRNA sequence. Extensive biochemical studies have shed light on the kinetics of this decoding process (reviewed in ref. 6). The aa-tRNA is delivered to the ribosome as part of a ternary complex (TC) with elongation factor thermo unstable (EF-Tu) and GTP. Initial binding of TC, mediated primarily by L7/L12 of the 50S subunit, is followed by the sampling of codon-anticodon interactions in the 30S A site. Codon-anticodon pairing leads to GTPase activation and GTP hydrolysis, which allows release of the acceptor end of aa-tRNA from EF-Tu. The aa-tRNA then either moves completely into the ribosomal A site (a step termed accommodation), where it can participate in peptide bond formation, or is rejected and released into solution...

Distinct functional classes oframmutations in 16S rRNA

“…Mutation G299A is located in h12, on the solvent side of the subunit near the S4/S5 interface (Fig. 1B,C), and destabilizes bridge B8 allosterically (Fagan et al 2013). All four of these mutations increase both missense and nonsense suppression in vivo (McClory et al 2010).…”

“…As mutations h8Δ3, h14Δ2, G347U, and G299A all cause defects in initial selection (McClory et al 2010;Fagan et al 2013), we tested whether the A-site ram mutations similarly affected EF-Tu-dependent GTP hydrolysis. Control and mutant 70S initiation complexes (70SICs), programmed with either a cognate UUU or near-cognate CUU codon in the A site, were rapidly mixed with EF-Tu•[γ 32 P]GTP•Phe-tRNA Phe under single-turnover conditions, and the reactions were quenched at various time points.…”

“…Mutations predicted to disrupt bridge B8 (i.e., h8Δ3, a 3-bp truncation of h8; h14Δ2, a 2-bp truncation of h14; and G347U in h14)-as well as mutation G299A in h12 -increase the rate of EF-Tu-dependent GTP hydrolysis, particularly in the near-cognate case. Crystal structures of mutant 70S ribosomes show that either G347U or G299A induces a distortion of h8/h14 that disrupts B8 (Fagan et al 2013). These conformational changes in h8/h14 are virtually identical to those seen in cocrystal structures of wild-type 70S ribosomes bound with TC (stabilized with either kirromycin or GDPCP) (Schmeing et al 2009;Voorhees et al 2010;Fagan et al 2013), suggesting that G347U and G299A act, at least in part, by promoting formation of the GTPase-activated state.…”

“…Crystal structures of mutant 70S ribosomes show that either G347U or G299A induces a distortion of h8/h14 that disrupts B8 (Fagan et al 2013). These conformational changes in h8/h14 are virtually identical to those seen in cocrystal structures of wild-type 70S ribosomes bound with TC (stabilized with either kirromycin or GDPCP) (Schmeing et al 2009;Voorhees et al 2010;Fagan et al 2013), suggesting that G347U and G299A act, at least in part, by promoting formation of the GTPase-activated state. Together, these observations indicate that disruption of bridge B8 is a critical aspect of GTPase activation and that h12 is allosterically linked to B8 (Fagan et al 2013).…”

“…Analysis of a subset of these 16S ram mutations has shown that bridge B8 increases the stringency of decoding by negatively regulating GTPase activation (McClory et al 2010;Fagan et al 2013). Mutations predicted to disrupt bridge B8 (i.e., h8Δ3, a 3-bp truncation of h8; h14Δ2, a 2-bp truncation of h14; and G347U in h14)-as well as mutation G299A in h12 -increase the rate of EF-Tu-dependent GTP hydrolysis, particularly in the near-cognate case.…”