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.

15 Reads
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Cell 07/2014; 158(1):121-31. DOI:10.1016/j.cell.2014.04.044 · 32.24 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Frontiers in Microbiology 04/2014; 5:170. DOI:10.3389/fmicb.2014.00170 · 3.99 Impact Factor
  • Source
    • "(center panels), where only the widths of each FRET state were constrained according to what is observed for DNA oligonucleotide standards imaged under identical conditions (Geggier et al., 2010). In so doing, the raw data was fit to three Gaussian distributions whose mean FRET values were 0.59, 0.41, and 0.26 (±0.01). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.
    Molecular cell 10/2011; 44(2):214-24. DOI:10.1016/j.molcel.2011.07.040 · 14.02 Impact Factor
Show more


15 Reads
Available from