Conformational Sampling of Aminoacyl-tRNA during Selection on the Bacterial Ribosome

Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 04/2010; 399(4):576-95. DOI: 10.1016/j.jmb.2010.04.038
Source: PubMed


Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.

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    • "Selection of the cognate tRNA has to proceed with optimal speed and accuracy. This is achieved by a complex, multistep pathway involving an initial selection step and a kinetic proofreading step (Rodnina and Wintermeyer, 2001; Geggier et al., 2010). Crucial for tRNA selection is the codon recognition step in the decoding center, and in particular the stabilization of codon-anticodon interaction by A-minor interactions with A1492/A1493 of 16S rRNA (A1824/A1825 in human) in the flipped-out conformation (Ogle et al., 2002). "
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    ABSTRACT: The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.
    Full-text · Article · Jul 2014 · Cell
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    • "During the trans-translation process, the alanyl-tmRNA/SmpB complex can enter the A site of the ribosome without the requirement for codon–anticodon interaction. The mechanisms involved in sense codon decoding in the canonical translation system have been well-characterized by kinetic experiments (Rodnina and Wintermeyer, 2001; Daviter et al., 2006), cryo-electron microscopic analyses (Stark et al., 2002; Valle et al., 2002, 2003a; Schuette et al., 2009; Villa et al., 2009), X-ray crystallographic analyses (Schmeing et al., 2009; Voorhees et al., 2010), and single-molecule observations (Blanchard et al., 2004; Lee et al., 2007; Geggier et al., 2010). These studies have identified a number of intermediate states of the sense codon decoding complex and have demonstrated that the selection of cognate aminoacyl-tRNA is achieved in two stages that are separated by irreversible GTP hydrolysis (Figure 4). "
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    ABSTRACT: During protein synthesis in cells, translating ribosomes may encounter abnormal situations that lead to retention of immature peptidyl-tRNA on the ribosome due to failure of suitable termination processes. Bacterial cells handle such situations by employing three systems that rescue the stalled translation machinery. The transfer messenger RNA/small protein B (tmRNA/SmpB) system, also called the trans-translation system, rescues stalled ribosomes by initiating template switching from the incomplete mRNA to the short open reading frame of tmRNA, leading to the production of a protein containing a C-terminal tag that renders it susceptible to proteolysis. The ArfA/RF2 and ArfB systems rescue stalled ribosomes directly by hydrolyzing the immature peptidyl-tRNA remaining on the ribosome. Here, the biochemical aspects of these systems, as clarified by recent studies, are reviewed.
    Full-text · Article · Apr 2014 · Frontiers in Microbiology
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    • "Thus, under the conditions of these experiments, the aa-tRNA selection process on T. thermophilus ribosomes operates with high fidelity, where the ternary complex rapidly dissociates during initial selection prior to GTPase activation and GTP hydrolysis (Blanchard et al. 2004;Geggier et al. 2010). In contrast, the binding of the ternary complex to ribosomes programmed with a cognate mRNA codon in the A-site in the presence of the nonhydrolyzable GTP analog GDPNP resulted in the formation of a relatively stable complex exhibiting intermediate-FRET, as expected from previous descriptions of the structural and kinetic features of the GTPase-activated (A/T) complex (Moazed et al. 1988;Pape et al. 2000;Blanchard et al. 2004;Geggier et al. 2010). In this state, aa-tRNA adopts a bent configuration in the A-site that is stabilized by domain closure of the small subunit and recognition of the minor groove of the mRNA codon–tRNA anticodon pair by residues A1492 and A1493 (Valle et al. 2003;Schmeing et al. 2009). "
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    ABSTRACT: The ribosome decodes mRNA by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16S rRNA nucleotides within the conformationally dynamic decoding site. By applying single-molecule FRET and X-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein S12 interfere with tRNA selection by allowing conformational distortions of the decoding site that impair GTPase activation of EF-Tu during the tRNA selection process. Distortions in the decoding site are reversed by streptomycin or by a second-site suppressor mutation in 16S rRNA. These observations encourage a refinement of the current model for decoding, wherein ribosomal protein S12 and the decoding site collaborate to optimize codon recognition and substrate discrimination during the early stages of the tRNA selection process.
    Full-text · Article · Oct 2013 · RNA
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