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1.
Faithful decoding of the genetic information by the ribosome relies on kinetically driven mechanisms that promote selection of cognate substrates during elongation. Recently, we have shown that in addition to these kinetically driven mechanisms, the ribosome possesses a post peptidyl transfer quality control system that retrospectively monitors the codon–anticodon interaction in the P site, triggering substantial losses in the specificity of the A site during subsequent tRNA and RF selection when a mistake has occurred. Here, we report a detailed kinetic analysis of tRNA selection in the context of a mismatched P-site codon:anticodon interaction. We observe pleiotropic effects of a P-site mismatch on tRNA selection, such that near-cognate tRNA is processed by the ribosome almost as efficiently as cognate. In particular, after a miscoding event, near-cognate codon–anticodon complexes are stabilized on the ribosome to an extent similar to that observed for cognate ones. Moreover, the two observed forward rates of GTPase activation and accommodation are greatly accelerated (∼10-fold) for near-cognate tRNAs. Because the ensemble of effects of a mismatched P site on substrate selection were found to be different from those reported for other ribosomal perturbations and miscoding agents, we propose that the structural integrity of the mRNA–tRNA helix in the P site provides a distinct molecular switch that dictates the specificity of the A site. 相似文献
2.
The ribosome must discriminate between correct and incorrect tRNAs with sufficient speed and accuracy to sustain an adequate rate of cell growth. Here, we report the results of explicit solvent molecular dynamics simulations, which address the mechanism of discrimination by the ribosome. The universally conserved 16S rRNA base A1493 and the kink in mRNA between A and P sites amplify differences in stability between cognate and near-cognate codon-anticodon pairs. Destabilization by the mRNA kink also provides a geometric explanation for the higher error rates observed for mismatches in the first codon position relative to mismatches in the second codon position. For more stable near-cognates, the repositioning of the universally conserved bases A1492 and G530 results in increased solvent exposure and an uncompensated loss of hydrogen bonds, preventing correct codon-anticodon-ribosome interactions from forming. 相似文献
3.
Wohlgemuth I Pohl C Mittelstaet J Konevega AL Rodnina MV 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2011,366(1580):2979-2986
Speed and accuracy of protein synthesis are fundamental parameters for the fitness of living cells, the quality control of translation, and the evolution of ribosomes. The ribosome developed complex mechanisms that allow for a uniform recognition and selection of any cognate aminoacyl-tRNA (aa-tRNA) and discrimination against any near-cognate aa-tRNA, regardless of the nature or position of the mismatch. This review describes the principles of the selection-kinetic partitioning and induced fit-and discusses the relationship between speed and accuracy of decoding, with a focus on bacterial translation. The translational machinery apparently has evolved towards high speed of translation at the cost of fidelity. 相似文献
4.
Accurate tRNA selection by the ribosome is essential for the synthesis of functional proteins. Previous structural studies indicated that the ribosome distinguishes between cognate and near-cognate tRNAs by monitoring the geometry of the codon–anticodon helix in the decoding center using the universally conserved 16S ribosomal RNA bases G530, A1492 and A1493. These bases form hydrogen bonds with the 2′-hydroxyl groups of the codon–anticodon helix, which are expected to be disrupted with a near-cognate codon–anticodon helix. However, a recent structural study showed that G530, A1492 and A1493 form hydrogen bonds in a manner identical with that of both cognate and near-cognate codon–anticodon helices. To understand how the ribosome discriminates between cognate and near-cognate tRNAs, we made 2′-deoxynucleotide and 2′-fluoro substituted mRNAs, which disrupt the hydrogen bonds between the A site codon and G530, A1492 and A1493. Our results show that multiple 2′-deoxynucleotide substitutions in the mRNA substantially inhibit tRNA selection, whereas multiple 2′-fluoro substitutions in the mRNA have only modest effects on tRNA selection. Furthermore, the miscoding antibiotics paromomycin and streptomycin rescue the defects in tRNA selection with the multiple 2′-deoxynucleotide substituted mRNA. These results suggest that steric complementarity in the decoding center is more important than the hydrogen bonds between the A site codon and G530, A1492 and A1493 for tRNA selection. 相似文献
5.
The speed and accuracy of protein synthesis are fundamental parameters for understanding the fitness of living cells, the quality control of translation, and the evolution of ribosomes. In this study, we analyse the speed and accuracy of the decoding step under conditions reproducing the high speed of translation in vivo. We show that error frequency is close to 10−3, consistent with the values measured in vivo. Selectivity is predominantly due to the differences in kcat values for cognate and near-cognate reactions, whereas the intrinsic affinity differences are not used for tRNA discrimination. Thus, the ribosome seems to be optimized towards high speed of translation at the cost of fidelity. Competition with near- and non-cognate ternary complexes reduces the rate of GTP hydrolysis in the cognate ternary complex, but does not appreciably affect the rate-limiting tRNA accommodation step. The GTP hydrolysis step is crucial for the optimization of both the speed and accuracy, which explains the necessity for the trade-off between the two fundamental parameters of translation. 相似文献
6.
The determination of diffusion coefficients from fluorescence recovery data is often complicated by geometric constraints imposed by the complex shapes of intracellular compartments. To address this issue, diffusion of proteins in the lumen of the endoplasmic reticulum (ER) is studied using cell biological and computational methods. Fluorescence recovery after photobleaching (FRAP) experiments are performed in tissue culture cells expressing GFP-KDEL, a soluble, fluorescent protein, in the ER lumen. The three-dimensional (3D) shape of the ER is determined by confocal microscopy and computationally reconstructed. Within these ER geometries diffusion of solutes is simulated using the method of particle strength exchange. The simulations are compared to experimental FRAP curves of GFP-KDEL in the same ER region. Comparisons of simulations in the 3D ER shapes to simulations in open 3D space show that the constraints imposed by the spatial confinement result in two- to fourfold underestimation of the molecular diffusion constant in the ER if the geometry is not taken into account. Using the same molecular diffusion constant in different simulations, the observed speed of fluorescence recovery varies by a factor of 2.5, depending on the particular ER geometry and the location of the bleached area. Organelle shape considerably influences diffusive transport and must be taken into account when relating experimental photobleaching data to molecular diffusion coefficients. This novel methodology combines experimental FRAP curves with high accuracy computer simulations of diffusion in the same ER geometry to determine the molecular diffusion constant of the solute in the particular ER lumen. 相似文献
7.
8.
During protein synthesis dictated by the codon sequence of messenger RNA, the ribosome selects aminoacyl-tRNA (aa-tRNA) with high accuracy, the exact mechanism of which remains elusive. By using a single-molecule fluorescence resonance energy transfer method coupled with fluorescence emission anisotropy, we provide evidence of random thermal motion of tRNAs within the ribosome in nanosecond timescale that we refer to as fluctuations. Our results indicate that cognate aa-tRNA fluctuates less frequently than near-cognate. This is counterintuitive because cognate aa-tRNA is expected to fluctuate more frequently to reach the ribosomal A-site faster than near-cognate. In addition, cognate aa-tRNA occupies the same position in the ribosome as near-cognate. These results argue for a mechanism which guides cognate aa-tRNA more accurately toward the A-site as compared to near-cognate. We suggest that a basis for this mechanism is the induced fit of the 30S subunit upon cognate aa-tRNA binding. Our single-molecule fluorescence resonance energy transfer time traces also point to a mechanistic model for GTP hydrolysis on elongation factor Tu mediated by aa-tRNA. 相似文献
9.
The fidelity of aminoacyl-tRNA (aa-tRNA) selection by the bacterial ribosome is determined by initial selection before and proofreading after GTP hydrolysis by elongation factor Tu. Here we report the rate constants of A-site binding of a near-cognate aa-tRNA. The comparison with the data for cognate aa-tRNA reveals an additional, important contribution to aa-tRNA discrimination of conformational coupling by induced fit. It is found that two rearrangement steps that limit the chemical reactions of A-site binding, i.e. GTPase activation (preceding GTP hydrolysis) and A-site accommodation (preceding peptide bond formation), are substantially faster for cognate than for near-cognate aa-tRNA. This suggests an induced-fit mechanism of aa-tRNA discrimination on the ribosome that operates in both initial selection and proofreading. It is proposed that the cognate codon-anticodon interaction, more efficiently than the near-cognate one, induces a particular conformation of the decoding center of 16S rRNA, which in turn promotes GTPase activation and A-site accommodation of aa-tRNA, thereby accelerating the chemical steps. As kinetically favored incorporation of the correct substrate has also been suggested for DNA and RNA polymerases, the present findings indicate that induced fit may contribute to the fidelity of template-programed systems in general. 相似文献
10.
Binding of aminoglycoside antibiotics to 16S ribosomal RNA induces a particular structure of the decoding center and increases the misincorporation of near-cognate amino acids. By kinetic analysis we show that this is due to stabilization of the near-cognate codon recognition complex and the acceleration of two rearrangements that limit the rate of amino acid incorporation. The same rearrangement steps are accelerated in the cognate coding situation. We suggest that cognate codon recognition, or near-cognate codon recognition augmented by aminoglycoside binding, promote the transition of 16S rRNA from a 'binding' to a 'productive' conformation that determines the fidelity of decoding. 相似文献
11.
The ribosome selects aminoacyl-tRNA (aa-tRNA) matching to the mRNA codon from the bulk of non-matching aa-tRNAs in two consecutive selection steps, initial selection and proofreading. Here we report the kinetic analysis of selection taking place under conditions where the overall selectivity was close to values observed in vivo and initial selection and proofreading contributed about equally. Comparison of the rate constants shows that the 350-fold difference in stabilities of cognate and near-cognate codon-anticodon complexes is not used for tRNA selection due to high rate of GTP hydrolysis in the cognate complex. tRNA selection at the initial selection step is entirely kinetically controlled and is due to much faster (650-fold) GTP hydrolysis of cognate compared to near-cognate substrate. 相似文献
12.
Aminoacyl-tRNAs (aa-tRNAs) are selected by the ribosome through a kinetically controlled induced fit mechanism. Cognate codon recognition induces a conformational change in the decoding center and a domain closure of the 30S subunit. We studied how these global structural rearrangements are related to tRNA discrimination by using streptomycin to restrict the conformational flexibility of the 30S subunit. The antibiotic stabilized aa-tRNA on the ribosome both with a cognate and with a near-cognate codon in the A site. Streptomycin altered the rates of GTP hydrolysis by elongation factor Tu (EF-Tu) on cognate and near-cognate codons, resulting in almost identical rates of GTP hydrolysis and virtually complete loss of selectivity. These results indicate that movements within the 30S subunit at the streptomycin-binding site are essential for the coupling between base pair recognition and GTP hydrolysis, thus modulating the fidelity of aa-tRNA selection. 相似文献
13.
Selection of tRNA by the ribosome requires a transition from an open to a closed form 总被引:21,自引:0,他引:21
A structural and mechanistic explanation for the selection of tRNAs by the ribosome has been elusive. Here, we report crystal structures of the 30S ribosomal subunit with codon and near-cognate tRNA anticodon stem loops bound at the decoding center and compare affinities of equivalent complexes in solution. In ribosomal interactions with near-cognate tRNA, deviation from Watson-Crick geometry results in uncompensated desolvation of hydrogen-bonding partners at the codon-anticodon minor groove. As a result, the transition to a closed form of the 30S induced by cognate tRNA is unfavorable for near-cognate tRNA unless paromomycin induces part of the rearrangement. We conclude that stabilization of a closed 30S conformation is required for tRNA selection, and thereby structurally rationalize much previous data on translational fidelity. 相似文献
14.
Selection of the correct start codon during initiation of translation on the ribosome is a key event in protein synthesis. In eukaryotic initiation, several factors have to function in concert to ensure that the initiator tRNA finds the cognate AUG start codon during mRNA scanning. The two initiation factors eIF1 and eIF1A are known to provide important functions for the initiation process and codon selection. Here, we have used molecular dynamics free energy calculations to evaluate the energetics of initiator tRNA binding to different near-cognate codons on the yeast 40S ribosomal subunit, in the presence and absence of these two initiation factors. The results show that eIF1 and eIF1A together cause a relatively uniform and high discrimination against near-cognate codons. This works such that eIF1 boosts the discrimination against a first position near-cognate G-U mismatch, and also against a second position A-A base pair, while eIF1A mainly acts on third codon position. The computer simulations further reveal the structural basis of the increased discriminatory effect caused by binding of eIF1 and eIF1A to the 40S ribosomal subunit. 相似文献
15.
Xiancheng Zeng Jeetender ChughAnette Casiano-Negroni Hashim M. Al-HashimiCharles L. Brooks III 《Journal of molecular biology》2014,426(19):3201-3213
Ribosomes control the missense error rate of ~ 10− 4 during translation though quantitative contributions of individual mechanistic steps of the conformational changes yet to be fully determined. Biochemical and biophysical studies led to a qualitative tRNA selection model in which ribosomal A-site residues A1492 and A1493 (A1492/3) flip out in response to cognate tRNA binding, promoting the subsequent reactions, but not in the case of near-cognate or non-cognate tRNA. However, this model was recently questioned by X-ray structures revealing conformations of extrahelical A1492/3 and domain closure of the decoding center in both cognate and near-cognate tRNA bound ribosome complexes, suggesting that the non-specific flipping of A1492/3 has no active role in tRNA selection. We explore this question by carrying out molecular dynamics simulations, aided with fluorescence and NMR experiments, to probe the free energy cost of extrahelical flipping of 1492/3 and the strain energy associated with domain conformational change. Our rigorous calculations demonstrate that the A1492/3 flipping is indeed a specific response to the binding of cognate tRNA, contributing 3 kcal/mol to the specificity of tRNA selection. Furthermore, the different A-minor interactions in cognate and near-cognate complexes propagate into the conformational strain and contribute another 4 kcal/mol in domain closure. The recent structure of ribosome with features of extrahelical A1492/3 and closed domain in near-cognate complex is reconciled by possible tautomerization of the wobble base pair in mRNA–tRNA. These results quantitatively rationalize other independent experimental observations and explain the ribosomal discrimination mechanism of selecting cognate versus near-cognate tRNA. 相似文献
16.
Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression. 相似文献
17.
Julian Rocha Jacqueline Corbitt Ting Yan Charles Richardson Andreas Gahlmann 《Biophysical journal》2019,116(10):1970-1983
The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in two-dimensional or three-dimensional single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions. 相似文献
18.
Ka-Weng Ieong Gabriele Indrisiunaite Arjun Prabhakar Joseph D Puglisi Mns Ehrenberg 《Nucleic acids research》2021,49(5):2684
We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km. 相似文献
19.
Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane. 相似文献
20.
Kojić N Huang A Chung E Ivanović M Filipović N Kojić M Tschumperlin DJ 《Biophysical journal》2010,99(11):3517-3525
Cells communicate through shed or secreted ligands that traffic through the interstitium. Force-induced changes in interstitial geometry can initiate mechanotransduction responses through changes in local ligand concentrations. To gain insight into the temporal and spatial evolution of such mechanotransduction responses, we developed a 3-D computational model that couples geometric changes observed in the lateral intercellular space (LIS) of mechanically loaded airway epithelial cells to the diffusion-convection equations that govern ligand transport. By solving the 3-D fluid field under changing boundary geometries, and then coupling the fluid velocities to the ligand transport equations, we calculated the temporal changes in the 3-D ligand concentration field. Our results illustrate the steady-state heterogeneities in ligand distribution that arise from local variations in interstitial geometry, and demonstrate that highly localized changes in ligand concentration can be induced by mechanical loading, depending on both local deformations and ligand convection effects. The occurrence of inhomogeneities at steady state and in response to mechanical loading suggest that local variations in ligand concentration may have important effects on cell-to-cell variations in basal signaling state and localized mechanotransduction responses. 相似文献