The role of fluctuations in tRNA selection by the ribosome

Abstract

The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is ''induced fit'' between the ribosome and tRNA. By using singlemolecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.ribosome dynamics ͉ ribosome selection ͉ single-molecule FRET T ranslation, protein synthesis by the ribosome, is a vital cellular process involving the two-subunit ribosomal particle, multiple RNAs, and protein cofactors (1, 2). To synthesize a protein molecule with correct sequence, the ribosome has to select correct transfer RNA (tRNA) as dictated by messenger RNA (mRNA). In the decoding site of the ribosome, the proper matching of the 3-nt codon sequence of the mRNA to the anticodon sequence of tRNA occurs at a rate of 20-30 per second with an error rate of 10 Ϫ4 (3).The difference in base-pairing energy between codon and anticodon cannot explain the low error rate of translation (4). In the past decades, considerable progress has been made in understanding the ingenious mechanisms used by the ribosome to achieve this low error rate (2, 4-6). The overall selection process consists of initial selection of the ternary complex [aminoacyl-tRNA (aa-tRNA), GTP, and elongation factor Tu (EF-Tu)], GTP hydrolysis in EF-Tu, and then proofreading (6). The GTP hydrolysis ensures that initial selection is separated from the proofreading by an irreversible reaction. This two-step selection yields overall probability of an error to be the product of the error probabilities of the two selection steps, thus dramatically lowering the overall chance of error (7).The initial selection of aa-tRNA consists of initial binding of the ternary complex on the ribosome, interaction between tRNA and mRNA (the codon recognition), and stabilization of the ternary complex onto the ribosome. GTPase activation takes place after additional stabilizing contacts are formed between the ribosome and the ternary complex (6,8). In the initial selection of aa-tRNA, the ternary complex binding induces structural changes in the ribosome (''induced-fit'') (5, 9) in which the 30S subunit of the ribosome changes its structure and forms additional stabilizing contacts with the incoming tRNA after the codon recognition (9). In case of one base-mismatch out of the 3 bp (near cognate), additional binding contacts are weaker, presumably because the mismatched bases form a less compact structure for the ribosome to ''wrap'' around as it forms additional induced fit contacts. In other words, the tighter binding of cognate tRNA relative to near-cognate tRNA mainly is attributable...

Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop

“…Recent work has demonstrated the utility of singlemolecule detection of fluorescent tRNAs in elucidating mechanistic details of tRNA interactions with the ribosome (Blanchard et al 2004a,b;Lee et al 2007;Munro et al 2007;Fei et al 2008). Although Zachau and his coworkers and others had demonstrated that several RNH 2 compounds (e.g., amines, hydrazines, hydrazides) could, in principle, be used to replace dihydroU within tRNA Zachau 1974, 1979;Yang and Söll 1974), as a practical manner, most studies of tRNA-ribosome interaction using this approach have focused virtually exclusively on proflavin-labeled tRNA (Rodnina et al 1994(Rodnina et al , 1997Pape et al 1998;Savelsbergh et al 2003;Pan et al 2006Pan et al , 2007Grigoriadou et al 2007a,b;Kothe and Rodnina 2007).…”

Stepwise loading of yeast clamp revealed by ensemble and single-molecule studies

“…We turned to a combination of single-molecule and ensemble methods to adequately identify the nature and steps anticipated along the PCNA loading-unloading pathways (27,28). Using primarily the FRET signals from a fluorescently labeled forked-DNA and PCNA, we obtained data that indicate (i) PCNA loading on DNA proceeds through a series of conformational intermediates and is successful after multiple failed attempts; (ii) RFC loads PCNA stoichiometrically and does not act catalytically on a primed 45-mer templated fork; (iii) the RFC · PCNA · DNA complex formed in the presence of ATP consists of at least two noninterconverting populations; (iv) the species of the RFC · PCNA · DNA complex disassemble either slowly through disassociation of the PCNA into its component subunits, a process that is independent of RFC and ATP/ATPγS levels, or through a more rapid recycling of intact PCNA; and (v) in the presence of polδ, a population of the RFC · PCNA · DNA complex is converted to the RFC · PCNA · DNA · polδ holoenzyme.…”

Models of Single-Molecule Experiments with Periodic Perturbations Reveal Hidden Dynamics in RNA Folding

“…3,5 This approach has been applied to monitor folding of biopolymers (proteins and nucleic acids), [7][8][9] enzyme-substrate complex formation, [10][11][12][13][14] and the operation of molecular motors. [15][16][17][18][19][20] A persistent issue in such studies is that they explicitly follow the dynamics of one, or at most a few, of the many degrees of freedom of a macromolecular system. In other words, the measurement is a projection of the full dynamics.…”