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1.
A conserved translation factor, known as EF-G in bacteria, promotes the translocation of tRNA and mRNA in the ribosome during protein synthesis. Here, EF-G.ribosome complexes in two intermediate states, before and after mRNA translocation, have been probed with hydroxyl radicals generated from free Fe(II)-EDTA. Before mRNA translocation and GTP hydrolysis, EF-G protected a limited set of nucleotides in both subunits of the ribosome from cleavage by hydroxyl radicals. In this state, an extensive set of nucleotides, in the platform and head domains of the 30S subunit and in the L7/L12 stalk region of the 50S subunit, became more exposed to hydroxyl radical attack, suggestive of conformational changes in these domains. Following mRNA translocation, EF-G protected a larger set of nucleotides (23S rRNA helices H43, H44, H89, and H95; 16S rRNA helices h5 and h15). No nucleotide with enhanced reactivity to hydroxyl radicals was detected in this latter state. Both before and after mRNA translocation, EF-G protected identical nucleotides in h5 and h15 of the 30S subunit. These results suggest that h5 and h15 may remain associated with EF-G during the dynamic course of the translocation mechanism. Nucleotides in H43 and H44 of the 50S subunit were protected only after translocation and GTP hydrolysis, suggesting that these helices interact dynamically with EF-G. The effects in H95 suggest that EF-G interacts weakly with H95 before mRNA translocation and strongly and more extensively with this helix following mRNA translocation.  相似文献   

2.
Prashant Khade 《FEBS letters》2010,584(2):420-7472
Recent X-ray crystal structures of the ribosome have revolutionized the field by providing a much-needed structural framework to understand ribosome function. Indeed, the crystal structures rationalize much of the genetic and biochemical data that have been meticulously gathered over 50 years. Here, we focus on the interactions between tRNAs and the ribosome and describe some of the insights that the structures provide about the mechanism of translation. Both high-resolution structures and functional studies are essential for fully appreciating the complex process of protein synthesis.  相似文献   

3.
In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.  相似文献   

4.
Translation of polyphenylalanine from a polyuridine template by the ribosome in the absence of the elongation factors EFG and EFTu (and the energy derived from GTP hydrolysis) is promoted by modification of the ribosome with thiol-specific reagents such as para-chloromercuribenzoate (pCMB). Here, we examine the translational cycle of modified ribosomes and show that peptide bond formation and tRNA binding are largely unaffected, whereas translocation of the mRNA:tRNA complex is substantially promoted by pCMB modification. The translocation movements that we observe are authentic by multiple criteria including the processivity of translation, accuracy of movement (three-nucleotide) along a defined mRNA template and sensitivity to antibiotics. Characterization of the modified ribosomes reveals that the protein content of the ribosomes is not depleted but that their subunit association properties are severely compromised. These data suggest that molecular targets (ribosomal proteins) in the interface region of the ribosome are critical barriers that influence the translocation of the mRNA:tRNA complex.  相似文献   

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7.
Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.  相似文献   

8.
The conformation of the anticodon stem-loop of tRNAs required for correct decoding by the ribosome depends on intramolecular and intermolecular interactions that are independent of the tRNA nucleotide sequence. Non-bridging phosphate oxygen atoms have been shown to be critical for the structure and function of several RNAs. However, little is known about the role they play in ribosomal A site binding and translocation of tRNA to the P site. Here, we show that non-bridging phosphate oxygen atoms within the tRNA anticodon stem-loop at positions 33, 35, and 37 are important for A site binding. Those at positions 34 and 36 are not necessary for binding, but are essential for translocation. Our results correlate with structural data, indicating that position 34 interacts with the highly conserved 16S rRNA base G966 and position 36 interacts with the universally conserved tRNA base U33 during translocation to the P site.  相似文献   

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10.
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   

11.
12.
Engaging the ribosome: universal IFs of translation   总被引:1,自引:0,他引:1  
Eukaryotic initiation factor 1A (eIF1A) and the GTPase IF2/eIF5B are the only universally conserved translation initiation factors. Recent structural, biochemical and genetic data indicate that these two factors form an evolutionarily conserved structural and functional unit in translation initiation. Based on insights gathered from studies of the translation elongation factor GTPases, we propose that these factors occupy the aminoacyl-tRNA site (A site) on the ribosome, and promote initiator tRNA binding and ribosomal subunit joining. These processes yield a translationally competent ribosome with Met-tRNA in the ribosomal peptidyl-tRNA site (P site), base-paired to the AUG start codon of a mRNA.  相似文献   

13.
We have trapped elongation factor G (EF-G) from Escherichia coli in six, functionally defined states, representing intermediates in its unidirectional catalytic cycle, which couples GTP hydrolysis to tRNA–mRNA translocation in the ribosome. By probing EF-G with trypsin in each state, we identified a substantial conformational change involving its conserved switch I (sw1) element, which contacts the GTP substrate. By attaching FeBABE (a hydroxyl radical generating probe) to sw1, we could monitor sw1 movement (by ∼20 Å), relative to the 70S ribosome, during the EF-G cycle. In free EF-G, sw1 is disordered, particularly in GDP-bound and nucleotide-free states. On EF-G•GTP binding to the ribosome, sw1 becomes structured and tucked inside the ribosome, thereby locking GTP onto EF-G. After hydrolysis and translocation, sw1 flips out from the ribosome, greatly accelerating release of GDP and EF-G from the ribosome. Collectively, our results support a central role of sw1 in driving the EF-G cycle during protein synthesis.  相似文献   

14.
Bacterial mRNAs are translated by closely spaced ribosomes and degraded from the 5′-end, with half-lives of around 2 min at 37 °C in most cases. Ribosome-free or “naked” mRNA is known to be readily degraded, but the initial event that inactivates the mRNA functionally has not been fully described. Here, we characterize a determinant of the functional stability of an mRNA, which is located in the early coding region. Using literature values for the mRNA half-lives of variant lacZ mRNAs in Escherichia coli, we modeled how the ribosome spacing is affected by the translation rate of the individual codons. When comparing the ribosome spacing at various segments of the mRNA to its functional half-life, we found a clear correlation between the functional mRNA half-life and the ribosome spacing in the mRNA region approximately between codon 20 and codon 45. From this finding, we predicted that inserts of slowly translated codons before codon 20 or after codon 45 should shorten or prolong, respectively, the functional mRNA half-life by altering the ribosome density in the important region. These predictions were tested on eight new lacZ variants, and their experimentally determined mRNA half-lives all supported the model. We thus suggest that translation-rate-mediated differences in the spacing between ribosomes in this early coding region is a parameter that determines the mRNAs functional half-life. We present a model that is in accordance with many earlier observations and that allows a prediction of the functional half-life of a given mRNA sequence.  相似文献   

15.
A key intermediate in translocation is an ‘unlocked state’ of the pre‐translocation ribosome in which the P‐site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two‐ and three‐colour smFRET imaging from multiple structural perspectives, EF‐G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF‐G‐bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.  相似文献   

16.
Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.  相似文献   

17.
The translocation step of protein elongation entails a large-scale rearrangement of the tRNA-mRNA-ribosome complex. Recent years have seen major advances in unraveling the mechanism of the process on the molecular level. A number of intermediate states have been defined and, in part, characterized structurally. The article reviews the recent evidence that suggests a dynamic role of the ribosome and its ligands during translocation. The focus is on dynamic aspects of tRNA movement and on the role of elongation factor G and GTP hydrolysis in translocation catalysis. The significance of structural changes of the ribosome induced by elongation factor G as well the role of ribosomal RNA are addressed. A functional model of elongation factor G as a motor protein driven by GTP hydrolysis is discussed.  相似文献   

18.
Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site bound codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5′-termini that could predetermine the position of the tRNAPhe on the ribosome by the P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3′ of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide—induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2–217) and/or in the C-terminal fragment 190–236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.  相似文献   

19.
细胞内部核糖体进入位点研究进展   总被引:2,自引:0,他引:2  
细胞内部核糖体进入位点(IRES)mRNA5’端非编码区的一段特殊的序列,它允许核糖体不从mRNA的5’到3’端阅读而直接在此序列处结合mRNA并起始翻译。本综述了IRES的发现、IRES的识别及细胞IRES的特征、作用机理、生物学意义及其生物学应用等方面的研究进展。  相似文献   

20.
We have used single‐particle reconstruction in cryo‐electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF‐Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF‐Tu, but before the release of EF‐Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 Å. Secondary structure elements in tRNA, EF‐Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF‐Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF‐Tu.  相似文献   

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