Distinct functions of elongation factor G in ribosome recycling and translocation

Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.     

Regulators of Cell Division in Plant Tissues

The cytokinins, 6-benzylaminopurine and kinetin, markedly enhanced the yield of both free and membrane-bound 80S ribosomes per unit weight of radish (Raphanus sativus) cotyledon tissue. The response was observed only after the induction of growth by cytokinin; during the lag period preceding cytokinin-induced growth, ribosome yields from both control and cytokinin-treated cotyledons were below detectable levels. Mannitol depressed both growth and ribosome yield to the same degree. The enhanced ribosome yield appeared to be an indirect effect of cytokinin and was probably a consequence of cytokinin-induced growth. The effect of 6-benzylaminopurine on ribosome yield was not reflected in enhanced levels of cytoplasmic ribosomal RNA, while recently synthesized ribosomes were found to be more readily recovered from cytokinin-treated tissue than from control tissue. It was concluded that cytokinin-enhanced ribosome yield resulted from enhanced ribosome recovery or extractability and that ribosome yield is an unreliable indication of ribosome level in plant tissue.     

Ribosome binding to inosine-substituted mRNAs in the absence of ATP and mRNA factors

Incubating ribosomes and eukaryotic initiation factor eIF3 with an inosine-substituted mRNA (where the mRNA secondary structure is strongly reduced) in the absence of ATP and other protein synthesis factors produces a 40 S ribosome.mRNA complex. When Met-tRNAMeti and eIF2 are added, a 60 S ribosome subunit attaches forming an 80 S ribosome.mRNA complex. ATP and the three mRNA factors, eIF4B, cap-site factor, and eIF4A, strongly stimulate the attachment of the 60 S subunit. In the absence of Met-tRNAMeti, the 60-S subunit does not attach, and adding ATP and the mRNA factors inhibits the accumulation of 40 S ribosome.inosine mRNA complexes. These results indicate that a 40 S ribosome, probably in a complex with eIF3, has an intrinsic capacity to attach to mRNA. Further, they suggest that Met-tRNAMeti may interact in a subsequent step to stabilize the 40 S ribosome.mRNA complex and allow the attachment of a 60 S ribosome subunit. Although seen most clearly with the inosine-substituted mRNAs, the 40 S ribosome reaction is also obtained with "guanosine" mRNA. A 40 S ribosome attaches to guanosine mRNA without ATP and mRNA factors when an incubation mixture containing ribosomes, eIF3, and mRNA is fixed with glutaraldehyde. In addition, a 40 S ribosome.guanosine mRNA complex can be obtained without glutaraldehyde in incubations containing ATP and the three mRNA factors in the absence of Met-tRNAMeti. The latter reaction is limited because of the instability of the 40 S ribosome.mRNA complex in the absence of Met-tRNA. Nevertheless, its authenticity is indicated by its full dependence upon ATP and the three mRNA factors. The lack of factor requirement for the formation of 40 S ribosome complexes with inosine-substituted mRNAs indicates that ATP and the three mRNA factors function primarily to unwind the secondary structure of a guanosine mRNA. Data relevant to a role for ATP in facilitating ribosome migration on an mRNA are also discussed.     

The ribosome as a molecular machine: the mechanism of tRNA-mRNA movement in translocation

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.     

On the rate of ribosome translocation anisotropy and ribosome saturation in the polysome

This study examines the rate of ribosome translocation in the mammalian polysome engaged in protein synthesis by utilizing our knowledge of the hydrodynamic behavior of the rat liver polysomes, sedimenting in a linear sucrose density gradient. The average distance between adjacent ribosomes in the polysome was estimated assuming an extended linear configuration of the polysomes during sedimentation. Based on this estimate, the velocity of ribosome movement along the messenger RNA appears to be non-uniform and inversely related to the ribosome content of the polysome. Such non-uniformity prevails at stages of translation prior to ribosome “saturation” of the polysome. A correlation has been made between the results reported herein and previously published evidence on the rate of polypeptide chain synthesis. The steady-state condition for the polypeptide chain assembly is viewed as representing the state of ribosome “saturation”, characterized by a minimal ribosome velocity and a maximum density of ribosome distribution, both functions being uniform throughout the entire length of the polysome.     

Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation

The journey of a newly synthesized polypeptide starts in the peptidyltransferase center of the ribosome, from where it traverses the exit tunnel. The interior of the ribosome exit tunnel is neither straight nor smooth. How the ribosome dynamics in vivo is influenced by the exit tunnel is poorly understood. Genome-wide ribosome profiling in mammalian cells reveals elevated ribosome density at the start codon and surprisingly the downstream 5th codon position as well. We found that the highly focused ribosomal pausing shortly after initiation is attributed to the geometry of the exit tunnel, as deletion of the loop region from ribosome protein L4 diminishes translational pausing at the 5th codon position. Unexpectedly, the ribosome variant undergoes translational abandonment shortly after initiation, suggesting that there exists an obligatory step between initiation and elongation commitment. We propose that the post-initiation pausing of ribosomes represents an inherent signature of the translation machinery to ensure productive translation.     

Insights into protein biosynthesis from structures of bacterial ribosomes

Understanding the structural basis of protein biosynthesis on the ribosome remains a challenging problem for cryo-electron microscopy and X-ray crystallography. Recent high-resolution structures of the Escherichia coli 70S ribosome without ligands, and of the Thermus thermophilus and E. coli 70S ribosomes with bound mRNA and tRNAs, reveal many new features of ribosome dynamics and ribosome-ligand interactions. In addition, the first high-resolution structures of the L7/L12 stalk of the ribosome, responsible for translation factor binding and GTPase activation, reveal the structural basis of the high degree of flexibility in this region of the ribosome. These structures provide groundbreaking insights into the mechanism of protein synthesis at the level of ribosome architecture, ligand binding and ribosome dynamics.     

Mechanism of fusidic acid inhibition of RRF- and EF-G-dependent splitting of the bacterial post-termination ribosome

The antibiotic drug fusidic acid (FA) is commonly used in the clinic against gram-positive bacterial infections. FA targets ribosome-bound elongation factor G (EF-G), a translational GTPase that accelerates both messenger RNA (mRNA) translocation and ribosome recycling. How FA inhibits translocation was recently clarified, but FA inhibition of ribosome recycling by EF-G and ribosome recycling factor (RRF) has remained obscure. Here we use fast kinetics techniques to estimate mean times of ribosome splitting and the stoichiometry of GTP hydrolysis by EF-G at varying concentrations of FA, EF-G and RRF. These mean times together with previous data on uninhibited ribosome recycling were used to clarify the mechanism of FA inhibition of ribosome splitting. The biochemical data on FA inhibition of translocation and recycling were used to model the growth inhibitory effect of FA on bacterial populations. We conclude that FA inhibition of translocation provides the dominant cause of bacterial growth reduction, but that FA inhibition of ribosome recycling may contribute significantly to FA-induced expression of short regulatory open reading frames, like those involved in FA resistance.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

Structure of the mammalian 80S ribosome at 8.7 A resolution

In this paper, we present a structure of the mammalian ribosome determined at approximately 8.7 A resolution by electron cryomicroscopy and single-particle methods. A model of the ribosome was created by docking homology models of subunit rRNAs and conserved proteins into the density map. We then modeled expansion segments in the subunit rRNAs and found unclaimed density for approximately 20 proteins. In general, many conserved proteins and novel proteins interact with expansion segments to form an integrated framework that may stabilize the mature ribosome. Our structure provides a snapshot of the mammalian ribosome at the beginning of translation and lends support to current models in which large movements of the small subunit and L1 stalk occur during tRNA translocation. Finally, details are presented for intersubunit bridges that are specific to the eukaryotic ribosome. We suggest that these bridges may help reset the conformation of the ribosome to prepare for the next cycle of chain elongation.     

RNase catalyzed hydrolysis of ribosomes in several functional states

The RNase A catalyzed hydrolysis of rRNA in ribosomes has been studied for nonwashed 50S and 70S ribosomes, for washed 50S and 70S ribosomes, for runoff 50S ribosomes and for 70S ribosomes in polysomes. The regions available to hydrolysis in the 50S ribosome remain available when the 50S ribosomes become a part of a 70S ribosome or a polysome. The regions available to hydrolysis in the 30S ribosome become unavailable when the 30S ribosome becomes part of a 70S ribosome or a polysome. Removal of tRNA, mRNA and factors from the 50S and 70S ribosome lowers the rate of hydrolysis of one site in the 23S rRNA. This shows that the conformation of one region of the 23S RNA changes for ribosomes in different functional states.     

核糖体展示口蹄疫单链抗体库的构建

目的:构建库容量大、多样性好的核糖体展示口蹄疫单链抗体(scFv)库。方法: 分离口蹄疫病毒免疫的兔脾细胞,提取总RNA,用RT-PCR扩增兔抗体的重链可变区(VH)基因和轻链可变区(VL)基因,同时扩增作为间隔区的兔抗体Ck基因;采用重叠延伸PCR (简称SOE-PCR)技术连接VH-VL基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接pMD18-T载体转化E.coli DH5α大肠杆菌,挑取阳性克隆测序以鉴定scFv组装。结果:成功构建了库容量达8.21×10¹³的兔源口蹄疫核糖体展示scFv库。结论: 构建的大容量兔源性口蹄疫核糖体展示抗体库可以成为进一步筛选特异性口蹄疫单链抗体的实验平台,为开发诊断性口蹄疫单链抗体奠定了很好的实验基础。     

Analysis of functioning ribosome content in Escherichia coli at different growth rates

Summary The amount of functioning ribosome in E. coli was measured using gel filtration chromatography. Cells were grown in a continuous fermentor to provide the same growth conditions at different growth rates. The functioning ribosome content and the fraction of functioning ribosome in the cell increased with growth rates. However, the nonfunctioning ribosome content was almost constant, regardless of the growth rate.     

Interaction strengths between the ribosome and tRNA at various steps of translocation

Transfer RNA (tRNA) translocates inside the ribosome during translation. We studied the interaction strengths between the ribosome and tRNA at various stages of translocation. We utilized an optical trap to measure the mechanical force to rupture tRNA from the ribosome. We measured the rupture forces of aminoacyl tRNA or peptidyl tRNA mimic from the ribosome in a prepeptidyl transfer state, the pretranslocational state, and the posttranslocational state. In addition, we measured the interaction strength between the ribosome and aminoacyl-tRNA in presence of viomycin. Based on the interaction strengths between the ribosome and tRNA under these conditions, 1), we concluded that tRNA interaction with the 30S subunit is far more important than the interaction with the 50S subunit in the mechanism of translocation; and 2), we propose a mechanism of translocation where the ribosomal ratchet motion, with the aid of EF-G, drives tRNA translocation.     

The binding of berenil to Escherichia coli ribosome.

The binding of the nonintercalating dye berenil to the 70S ribosome of Escherichia coli has been demonstrated by spectrophotometric measurements and gel filtration through Biogel P100 column. The berenil spectrum is gradually shifted towards the red region with the increasing amount of ribosome added, the isosbestic point being at 375 nm. There is positive cooperativity in the binding of berenil to the ribosome as demonstrated by the equilibrium dialysis. On binding with berenil, the ribosome is degraded faster by RNase I especially at low Mg++ concentration and its capacity to inhibit RNase I catalysed hydrolysis of ribopolymers is decreased. These indicate the unfolding of the structure of the ribosome.     

Growth phase-coupled changes of the ribosome profile in natural isolates and laboratory strains of Escherichia coli

The growth phase-dependent change in sucrose density gradient centrifugation patterns of ribosomes was analyzed for both laboratory strains of Escherichia coli and natural isolates from the ECOR collection. All of the natural isolates examined formed 100S ribosome dimers in the stationary phase, and ribosome modulation factor (RMF) was associated with the ribosome dimers in the ECOR strains as in the laboratory strain W3110. The ribosome profile (70S monomers versus 100S dimers) follows a defined pattern over time during lengthy culture in both the laboratory strains and natural isolates. There are four discrete stages: (i) formation of 100S dimers in the early stationary phase; (ii) transient decrease in the dimer level; (iii) return of dimers to the maximum level; and (iv) dissociation of 100S dimers into 70S ribosomes, which are quickly degraded into subassemblies. The total time for this cycle of ribosome profile change, however, varied from strain to strain, resulting in apparent differences in the ribosome profiles when observed at a fixed time point. A correlation was noted in all strains between the decay of 100S ribosomes and the subsequent loss of cell viability. Two types of E. coli mutants defective in ribosome dimerization were identified, both of which were unable to survive for a prolonged period in stationary phase. The W3110 mutant, with a disrupted rmf gene, has a defect in ribosome dimerization because of lack of RMF, while strain Q13 is unable to form ribosome dimers due to a ribosomal defect in binding RMF.     

Stepwise Splitting of Ribosomal Proteins from Yeast Ribosomes by LiCl

Structural studies have revealed that the core of the ribosome structure is conserved among ribosomes of all kingdoms. Kingdom-specific ribosomal proteins (r-proteins) are located in peripheral parts of the ribosome. In this work, the interactions between rRNA and r-proteins of eukaryote Saccharomyces cerevisiae ribosome were investigated applying LiCl induced splitting and quantitative mass spectrometry. R-proteins were divided into four groups according to their binding properties to the rRNA. Most yeast r-proteins are removed from rRNA by 0.5–1 M LiCl. Eukaryote-specific r-proteins are among the first to dissociate. The majority of the strong binders are known to be required for the early ribosome assembly events. As compared to the bacterial ribosome, yeast r-proteins are dissociated from rRNA at lower ionic strength. Our results demonstrate that the nature of protein-RNA interactions in the ribosome is not conserved between different kingdoms.     

Directed evolution of the lambda receptor of Escherichia coli through affinity chromatographic selection

Two low-resolution three-dimensional maps of the structure of crystalline ribosomes from the oocytes of the lizard, Lacerta sicula, have been obtained by electron microscopy and image processing. One map, derived from sheets contrasted with gold-thioglucose, shows the whole ribosome in outline. The other map, based on sheets embedded in glucose, shows predominantly the RNA in the ribosome.The distribution of RNA-rich and protein-rich regions within the ribosome was assessed by comparing both maps. The RNA forms a dense central core, while the ribosomal protein is located mainly at the periphery and constitutes most of the ribosome surface. The RNA appears to be accessible at several sites on the surface. The two subunits of the ribosome are not resolved, indicating that they are in close contact with one another. The subunit interface cuts through a region of the ribosome that is particularly rich in RNA.     

从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。     

Single-molecule study of ribosome hierarchic dynamics at the peptidyl transferase center

During protein biosynthesis the ribosome moves along mRNA in steps of precisely three nucleotides. The mechanism for this ribosome motion remains elusive. Using a classification algorithm to sort single-molecule fluorescence resonance energy transfer data into subpopulations, we found that the ribosome dynamics detected at the peptidyl transferase center are highly inhomogeneous. The pretranslocation complex has at least four subpopulations that sample two hybrid states, whereas the posttranslocation complex is mainly static. We observed transitions among the ribosome subpopulations under various conditions, including 1), in the presence of EF-G; 2), spontaneously; 3), in different buffers, and 4), bound to antibiotics. Therefore, these subpopulations represent biologically active ribosomes. One key observation indicates that the Hy2 hybrid state only exists in a fluctuating ribosome subpopulation, which prompts us to propose that ribosome dynamics are hierarchically arranged. This proposal may have important implications for the regulation of cellular translation rates.