Crystal structure of the eukaryotic ribosome

Crystal structures of prokaryotic ribosomes have described in detail the universally conserved core of the translation mechanism. However, many facets of the translation process in eukaryotes are not shared with prokaryotes. The crystal structure of the yeast 80S ribosome determined at 4.15 angstrom resolution reveals the higher complexity of eukaryotic ribosomes, which are 40% larger than their bacterial counterparts. Our model shows how eukaryote-specific elements considerably expand the network of interactions within the ribosome and provides insights into eukaryote-specific features of protein synthesis. Our crystals capture the ribosome in the ratcheted state, which is essential for translocation of mRNA and transfer RNA (tRNA), and in which the small ribosomal subunit has rotated with respect to the large subunit. We describe the conformational changes in both ribosomal subunits that are involved in ratcheting and their implications in coordination between the two associated subunits and in mRNA and tRNA translocation.     

Structures of the bacterial ribosome in classical and hybrid states of tRNA binding

During protein synthesis, the ribosome controls the movement of tRNA and mRNA by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of ~3.2 angstroms by means of x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.     

Visualizing ribosome biogenesis: parallel assembly pathways for the 30S subunit

Ribosomes are self-assembling macromolecular machines that translate DNA into proteins, and an understanding of ribosome biogenesis is central to cellular physiology. Previous studies on the Escherichia coli 30S subunit suggest that ribosome assembly occurs via multiple parallel pathways rather than through a single rate-limiting step, but little mechanistic information is known about this process. Discovery single-particle profiling (DSP), an application of time-resolved electron microscopy, was used to obtain more than 1 million snapshots of assembling 30S subunits, identify and visualize the structures of 14 assembly intermediates, and monitor the population flux of these intermediates over time. DSP results were integrated with mass spectrometry data to construct the first ribosome-assembly mechanism that incorporates binding dependencies, rate constants, and structural characterization of populated intermediates.     

Micelles protect membrane complexes from solution to vacuum

The ability to maintain interactions between soluble protein subunits in the gas phase of a mass spectrometer gives critical insight into the stoichiometry and interaction networks of protein complexes. Conversely, for membrane protein complexes in micelles, the transition into the gas phase usually leads to the disruption of interactions, particularly between cytoplasmic and membrane subunits, and a mass spectrum dominated by large aggregates of detergent molecules. We show that by applying nanoelectrospray to a micellar solution of a membrane protein complex, the heteromeric adenosine 5'-triphosphate (ATP)-binding cassette transporter BtuC2D2, we can maintain the complex intact in the gas phase of a mass spectrometer. Dissociation of either transmembrane (BtuC) or cytoplasmic (BtuD) subunits uncovers modifications to the transmembrane subunits and cooperative binding of ATP. By protecting a membrane protein complex within a n-dodecyl-beta-d-maltoside micelle, we demonstrated a powerful strategy that will enable the subunit stoichiometry and ligand-binding properties of membrane complexes to be determined directly, by precise determination of the masses of intact complexes and dissociated subunits.     

Small-molecule inhibition of TNF-alpha

We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.     

RNA structure: reading the ribosome

The crystal structures of the ribosome and its subunits have increased the amount of information about RNA structure by about two orders of magnitude. This is leading to an understanding of the principles of RNA folding and of the molecular interactions that underlie the functional capabilities of the ribosome and other RNA systems. Nearly all of the possible types of RNA tertiary interactions have been found in ribosomal RNA. One of these, an abundant tertiary structural motif called the A-minor interaction, has been shown to participate in both aminoacyl-transfer RNA selection and in peptidyl transferase; it may also play an important role in the structural dynamics of the ribosome.     

Structural basis for the rescue of stalled ribosomes: structure of YaeJ bound to the ribosome

In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA(i)(fMet) and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.     

核糖体的结构与功能研究

从核糖体功能中长期存在之谜出发,阐述了关于核糖体亚基高分辨率晶体结构的研究,分析了核糖体结构与mRNA转录精确性的关系,及肽键形成机制与50S亚基结构的关系,论述了核糖体亚基结构在抗生素研制中的应用,并对核糖体功能复合物晶体结构研究的新方向进行了展望。     

Crystal structure of the ribosome at 5.5 A resolution

We describe the crystal structure of the complete Thermus thermophilus 70S ribosome containing bound messenger RNA and transfer RNAs (tRNAs) at 5.5 angstrom resolution. All of the 16S, 23S, and 5S ribosomal RNA (rRNA) chains, the A-, P-, and E-site tRNAs, and most of the ribosomal proteins can be fitted to the electron density map. The core of the interface between the 30S small subunit and the 50S large subunit, where the tRNA substrates are bound, is dominated by RNA, with proteins located mainly at the periphery, consistent with ribosomal function being based on rRNA. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. The tRNAs are closely juxtaposed with the intersubunit bridges, in a way that suggests coupling of the 20 to 50 angstrom movements associated with tRNA translocation with intersubunit movement.     

Distribution of protein and RNA in the 30S ribosomal subunit

In Escherichia coli, the small ribosomal subunit has a sedimentation coefficient of 30S, and consists of a 16S RNA molecule of 1541 nucleotides complexed with 21 proteins. Over the last few years, a controversy has emerged regarding the spatial distribution of RNA and protein in the 30S subunit. Contrast variation with neutron scattering was used to suggest that the RNA was located in a central core of the subunit and the proteins mainly in the periphery, with virtually no separation between the centers of mass of protein and RNA. However, these findings are incompatible with the results of efforts to locate individual ribosomal proteins by immune electron microscopy and triangulation with interprotein distance measurements. The conflict between these two views is resolved in this report of small-angle neutron scattering measurements on 30S subunits with and without protein S1, and on subunits reconstituted from deuterated 16S RNA and unlabeled proteins. The results show that (i) the proteins and RNA are intermingled, with neither component dominating at the core or the periphery, and (ii) the spatial distribution of protein and RNA is asymmetrical, with a separation between their centers of mass of about 25 angstroms.     

Composition and Content of High-Molecular-Weight Glutenin Subunits and Their Effects on Wheat Quality

Sedimentation values, flour glutenin macropolymer (GMP) contents, composition and contents of high-molecular-weight (HMW) glutenin subunits (GS) of 233 flour samples were determined. Our data indicated that subunit 1 occurred more frequently at Glu-A1, subunit pair 7 + 8 at Glu- B1 and 2 + 12 at Glu-D1. The significant relationships between Glu-1 quality score and total HMW glutenin content, sedimentation value and GMP content suggested that the composition of HMW-GS affects wheat quality strongly. Moreover,the total content of HMW-GS was correlated with certain quality parameters more significantly. Relationship between subunit 5 + 10 content and breadmaking quality was better than others, but 2 + 12, 7 + 8, 7 + 9 and 4 + 12 also correlated with certain quality parameters significantly. The contents of total HMW-glutenin, x-type subunits and y-type subunits related with sedimentation value, flour GMP content, and Glu-1 quality score more strongly than that of individual subunit or subunit pair. The flour GMP content, with excellent correlation to sedimentation value, total contents of HMW glutenin, x- and y-type subunits and many other quality parameters, could be an ideal indicator of breadmaking quality at earlier generations for breeding purpose for its simple procedure and small scale.     

高分子量谷蛋白亚基组成及其含量与小麦品质关系研究

 测定了国内外 2 33份小麦面粉样品的沉降值、谷蛋白大聚体 (GMP)含量及其高分子量谷蛋白亚基(HMW GS)组成与含量。结果表明 ,国内小麦品种 (系 )A1位点亚基 1出现的频率最高 ,而亚基 2 出现的频率较低 ;B1位点 7+8亚基对出现的频率最高 ,其次是 7+9亚基对 ;D1位点 2 +12亚基对出现的频率高于其它亚基对。亚基组成与品质关系密切 ,亚基评分与HMW谷蛋白总量、沉降值和GMP含量的相关均达到极显著水平。HMW GS含量与小麦品质相关性更高 ,5 +10亚基对含量与品质的相关性明显高于其它亚基对 ,2 +12、7+8、7+9、4 +12亚基对含量与品质的相关性也达到极显著水平。GMP含量测量简单 ,用样量少 ,且与沉降值、HMW谷蛋白总量、X 型、Y 型亚基含量和多种亚基及亚基对含量呈显著正相关 ,可作为小麦品质早代选择的指标。     

密穗小麦(Triticum compactum)Glu-1位点亚基组成分析

采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对来自21个国家的50份密穗小麦Glu-1位点亚基组成进行了鉴定与分析.结果表明,在3个位点共检测到15种不同的亚基类型,21种组合形式.其中,以2*,7 8,2 12和N,7 8,2 12组合为主,各占22%和16%.在各位点等位变异中,Glu-B1位点变异类型最多,共10种,以7 8(42%)、20(20%)和21(12%)为主.Glu-A1和Glu-D1位点各有3种和2种等位变异,分别以N(50%)和2 12(88%)为优势亚基.更为重要的是选出一些具有优质亚基的材料,其中1份材料亚基组合为(1、7 8、5 10),品质评分达到满分(10分)可供小麦遗传改良.     

How hibernation factors RMF, HPF, and YfiA turn off protein synthesis

Eubacteria inactivate their ribosomes as 100S dimers or 70S monomers upon entry into stationary phase. In Escherichia coli, 100S dimer formation is mediated by ribosome modulation factor (RMF) and hibernation promoting factor (HPF), or alternatively, the YfiA protein inactivates ribosomes as 70S monomers. Here, we present high-resolution crystal structures of the Thermus thermophilus 70S ribosome in complex with each of these stationary-phase factors. The binding site of RMF overlaps with that of the messenger RNA (mRNA) Shine-Dalgarno sequence, which prevents the interaction between the mRNA and the 16S ribosomal RNA. The nearly identical binding sites of HPF and YfiA overlap with those of the mRNA, transfer RNA, and initiation factors, which prevents translation initiation. The binding of RMF and HPF, but not YfiA, to the ribosome induces a conformational change of the 30S head domain that promotes 100S dimer formation.     

Structural roles for human translation factor eIF3 in initiation of protein synthesis

Protein synthesis in mammalian cells requires initiation factor eIF3, a approximately 750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5'-cap or an internal ribosome entry site (IRES). Cryo-electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5'-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.     

Nicotinic Acetylcholine Receptor Gene Family of the Pea Aphid,Acyrthosiphon pisum

The nicotinic acetylcholine receptors （nAchRs） are cholinergic receptors that form ligand-gated ion channels by ifve subunits in insect and vertebrate nervous systems. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Here, we identiifed and cloned 11 candidate nAChR subunit genes in Acyrthosiphon pisum using genome-based bioinformatics combined modern molecular techniques. Most A. pisum nAChRs including α1, α2, α3, α4, α6, α8, and β1 show highly sequence identities with the counterparts of other insects examined. Expression proifles analysis showed that all subunit genes were expressed in adult head. At least two subunits have alternative splicing that obviously increase A. pisum nicotinic receptor diversity. This study will be invaluable for exploring the molecular mechanisms of neonicotinoid-like insecticides in sucking pests, and for ultimately establishing the screening platform of novel insecticides.     

马卡小麦高分子谷蛋白亚基遗传变异分析

利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)方法对29份马卡小麦材料进行了高分子谷蛋白亚基(HMW-GS)分析。结果表明,供试材料中存在着较丰富的亚基变异,共检测到9种亚基类型和9种亚基组合。其中,Glu-A1位点上null亚基出现频率最高,达82·8%。Glu-B1位点上7+8为优势亚基(占53·3%),7+9亚基出现频率也较高23·3%。Glu-D1位点上优势亚基为2+12(占82·8%)。供试材料品质评分变幅为4~8分,平均为6·2分。     

拟南芥复制因子C亚基3基因突变削弱了植物对紫外辐射的抗性

在前期以EMS (甲基磺酸乙酯)筛选更多抗生物和非生物逆境的突变体试验中,发现了1个从G到A的点突变体rfc3-1,该点突变削弱了植株抗紫外辐射的能力.将野生型拟南芥复制因子C亚基3基因转化到突变株rfc3-1后恢复了突变株的野生型表型,证明紫外辐射抗性异常表型是由拟南芥复制因子C亚基3基因突变所引起的,AtRFC3在拟南芥紫外辐射抗性中起重要作用.     

Isolation and Sequence Analysis of HMW Glutenin Subunit 1Dy10.1 Ecoding Gene from Xinjiang Wheat (Triticum petropavlovskyi Udacz.et Migusch)

A novel HMW glutenin subunit gene 1Dy10.1 was isolated and characterized from Xinjiang wheat (Triticum petropavlovskyi. Udacz. et Migusch) accession Daomai 2. The complete open reading frame (ORF) of 1Dy10.1 was 1965 bp, encoding 655 amino acids. The numbers and distribution of cysteines in 1Dy10.1 were similar to those of 1Dy10 and other y-type subunits. In the N-terminal of 1Dy10.1, an amino acid was changed from L (leucine) to P (proline) at position 55. The repetitive domain of 1Dy10.1 differed from those of known HMW subunits by substitutions, insertions or/and deletions involving single or more amino acid residues. In the repetitive domain of subunit 1Dy10.1, the deletion of tripeptide GQQ in the consensus unit PGQGQQ resulted in the appearance of the motif PGQ that have not been observed in other known y-type HMW subunits. In comparison with the subunit 1Dy12, a deletion of dipeptide GQ, which occurred in subunit 1Dy10, was also observed in subunit 1Dy10.1. The cloned 1Dyl0.1 gene had been successfully expressed in Escherichia coli, and the expressed protein had the identical mobility with the endogenous subunit 1Dyl0.1 from seed.     

Structure of the rotor ring of F-Type Na+-ATPase from Ilyobacter tartaricus

In the crystal structure of the membrane-embedded rotor ring of the sodium ion-translocating adenosine 5'-triphosphate (ATP) synthase of Ilyobacter tartaricus at 2.4 angstrom resolution, 11 c subunits are assembled into an hourglass-shaped cylinder with 11-fold symmetry. Sodium ions are bound in a locked conformation close to the outer surface of the cylinder near the middle of the membrane. The structure supports an ion-translocation mechanism in the intact ATP synthase in which the binding site converts from the locked conformation into one that opens toward subunit a as the rotor ring moves through the subunit a/c interface.