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.     

Decoding in the absence of a codon by tmRNA and SmpB in the ribosome

In bacteria, ribosomes stalled at the end of truncated messages are rescued by transfer-messenger RNA (tmRNA), a bifunctional molecule that acts as both a transfer RNA (tRNA) and a messenger RNA (mRNA), and SmpB, a small protein that works in concert with tmRNA. Here, we present the crystal structure of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome at 3.2 angstroms resolution. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome and explains why the tmRNA-SmpB system does not interfere with normal translation.     

Impaired control of IRES-mediated translation in X-linked dyskeratosis congenita

The DKC1 gene encodes a pseudouridine synthase that modifies ribosomal RNA (rRNA). DKC1 is mutated in people with X-linked dyskeratosis congenita (X-DC), a disease characterized by bone marrow failure, skin abnormalities, and increased susceptibility to cancer. How alterations in ribosome modification might lead to cancer and other features of the disease remains unknown. Using an unbiased proteomics strategy, we discovered a specific defect in IRES (internal ribosome entry site)-dependent translation in Dkc1(m) mice and in cells from X-DC patients. This defect results in impaired translation of messenger RNAs containing IRES elements, including those encoding the tumor suppressor p27(Kip1) and the antiapoptotic factors Bcl-xL and XIAP (X-linked Inhibitor of Apoptosis Protein). Moreover, Dkc1(m) ribosomes were unable to direct translation from IRES elements present in viral messenger RNAs. These findings reveal a potential mechanism by which defective ribosome activity leads to disease and cancer.     

Dom34:Hbs1 promotes subunit dissociation and peptidyl-tRNA drop-off to initiate no-go decay

No-go decay (NGD) is one of several messenger RNA (mRNA) surveillance systems dedicated to the removal of defective mRNAs from the available pool. Two interacting factors, Dom34 and Hbs1, are genetically implicated in NGD in yeast. Using a reconstituted yeast translation system, we show that Dom34:Hbs1 interacts with the ribosome to promote subunit dissociation and peptidyl-tRNA drop-off. Our data further indicate that these recycling activities are shared by the homologous translation termination factor complex eRF1:eRF3, suggesting a common ancestral function. Because Dom34:Hbs1 activity exhibits no dependence on either peptide length or A-site codon identity, we propose that this quality-control system functions broadly to recycle ribosomes throughout the translation cycle whenever stalls occur.     

Ribosome assembly factors prevent premature translation initiation by 40S assembly intermediates

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs) and occurs through ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here, we present the electron cryo-microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae at 18 angstrom resolution. We obtained cryo-EM reconstructions of preribosomal complexes lacking individual components to define the positions of all seven AFs bound to this intermediate. These late-binding AFs are positioned to prevent each step in the translation initiation pathway. Together, they obstruct the binding sites for initiation factors, prevent the opening of the messenger RNA channel, block 60S subunit joining, and disrupt the decoding site. These redundant mechanisms probably ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation.     

A probabilistic functional network of yeast genes

A conceptual framework for integrating diverse functional genomics data was developed by reinterpreting experiments to provide numerical likelihoods that genes are functionally linked. This allows direct comparison and integration of different classes of data. The resulting probabilistic gene network estimates the functional coupling between genes. Within this framework, we reconstructed an extensive, high-quality functional gene network for Saccharomyces cerevisiae, consisting of 4681 (approximately 81%) of the known yeast genes linked by approximately 34,000 probabilistic linkages comparable in accuracy to small-scale interaction assays. The integrated linkages distinguish true from false-positive interactions in earlier data sets; new interactions emerge from genes' network contexts, as shown for genes in chromatin modification and ribosome biogenesis.     

Translational operator of mRNA on the ribosome: how repressor proteins exclude ribosome binding

The ribosome of Thermus thermophilus was cocrystallized with initiator transfer RNA (tRNA) and a structured messenger RNA (mRNA) carrying a translational operator. The path of the mRNA was defined at 5.5 angstroms resolution by comparing it with either the crystal structure of the same ribosomal complex lacking mRNA or with an unstructured mRNA. A precise ribosomal environment positions the operator stem-loop structure perpendicular to the surface of the ribosome on the platform of the 30S subunit. The binding of the operator and of the initiator tRNA occurs on the ribosome with an unoccupied tRNA exit site, which is expected for an initiation complex. The positioning of the regulatory domain of the operator relative to the ribosome elucidates the molecular mechanism by which the bound repressor switches off translation. Our data suggest a general way in which mRNA control elements must be placed on the ribosome to perform their regulatory task.     

Signal recognition particle receptor exposes the ribosomal translocon binding site

Signal sequences of secretory and membrane proteins are recognized by the signal recognition particle (SRP) as they emerge from the ribosome. This results in their targeting to the membrane by docking with the SRP receptor, which facilitates transfer of the ribosome to the translocon. Here, we present the 8 angstrom cryo-electron microscopy structure of a "docking complex" consisting of a SRP-bound 80S ribosome and the SRP receptor. Interaction of the SRP receptor with both SRP and the ribosome rearranged the S domain of SRP such that a ribosomal binding site for the translocon, the L23e/L35 site, became exposed, whereas Alu domain-mediated elongation arrest persisted.     

Ribosome profiling shows that miR-430 reduces translation before causing mRNA decay in zebrafish

MicroRNAs regulate gene expression through deadenylation, repression, and messenger RNA (mRNA) decay. However, the contribution of each mechanism in non-steady-state situations remains unclear. We monitored the impact of miR-430 on ribosome occupancy of endogenous mRNAs in wild-type and dicer mutant zebrafish embryos and found that miR-430 reduces the number of ribosomes on target mRNAs before causing mRNA decay. Translational repression occurs before complete deadenylation, and disrupting deadenylation with use of an internal polyadenylate tail did not block target repression. Lastly, we observed that ribosome density along the length of the message remains constant, suggesting that translational repression occurs by reducing the rate of initiation rather than affecting elongation or causing ribosomal drop-off. These results show that miR-430 regulates translation initiation before inducing mRNA decay during zebrafish development.     

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.     

The mechanism for activation of GTP hydrolysis on the ribosome

Protein synthesis requires several guanosine triphosphatase (GTPase) factors, including elongation factor Tu (EF-Tu), which delivers aminoacyl-transfer RNAs (tRNAs) to the ribosome. To understand how the ribosome triggers GTP hydrolysis in translational GTPases, we have determined the crystal structure of EF-Tu and aminoacyl-tRNA bound to the ribosome with a GTP analog, to 3.2 angstrom resolution. EF-Tu is in its active conformation, the switch I loop is ordered, and the catalytic histidine is coordinating the nucleophilic water in position for inline attack on the γ-phosphate of GTP. This activated conformation is due to a critical and conserved interaction of the histidine with A2662 of the sarcin-ricin loop of the 23S ribosomal RNA. The structure suggests a universal mechanism for GTPase activation and hydrolysis in translational GTPases on the ribosome.     

基于颜色直方图和LBP-TD算子的木板材节疤缺陷区域检测

提出一种基于颜色和纹理信息的木板材表面节疤缺陷区域检测方法。首先,根据木板材表面图像中正常区域和缺陷区域的颜色差异,通过颜色直方图自动获取缺陷区域的种子点;然后,提出一种纹理扩散算法,它从种子点出发,基于图像局部纹理特征搜索缺陷区域的边缘。此外,改进了局部二进制模式算子,提出一种LBP-TD算子以更好地适应纹理扩散。实验结果表明:针对各种常见的木板材节疤缺陷,当缺陷区域与正常木纹区域的颜色、纹理存在较明显差异时,无论木纹本身是否规则,本文方法都能准确地检测出木板材节疤缺陷的区域;而当缺陷区域与正常木纹区域的颜色、纹理的差异均不明显时,本文方法仍能检测出缺陷区域的大致轮廓。数据对比显示了本文方法的误检率要低于传统的OTSU法。      

An active role for tRNA in decoding beyond codon:anticodon pairing

During transfer RNA (tRNA) selection, a cognate codon:anticodon interaction triggers a series of events that ultimately results in the acceptance of that tRNA into the ribosome for peptide-bond formation. High-fidelity discrimination between the cognate tRNA and near- and noncognate ones depends both on their differential dissociation rates from the ribosome and on specific acceleration of forward rate constants by cognate species. Here we show that a mutant tRNA(Trp) carrying a single substitution in its D-arm achieves elevated levels of miscoding by accelerating these forward rate constants independent of codon:anticodon pairing in the decoding center. These data provide evidence for a direct role for tRNA in signaling its own acceptance during decoding and support its fundamental role during the evolution of protein synthesis.     

Insights into translational termination from the structure of RF2 bound to the ribosome

The termination of protein synthesis occurs through the specific recognition of a stop codon in the A site of the ribosome by a release factor (RF), which then catalyzes the hydrolysis of the nascent protein chain from the P-site transfer RNA. Here we present, at a resolution of 3.5 angstroms, the crystal structure of RF2 in complex with its cognate UGA stop codon in the 70S ribosome. The structure provides insight into how RF2 specifically recognizes the stop codon; it also suggests a model for the role of a universally conserved GGQ motif in the catalysis of peptide release.     

How to make water run uphill

A surface having a spatial gradient in its surface free energy was capable of causing drops of water placed on it to move uphill. This motion was the result of an imbalance in the forces due to surface tension acting on the liquid-solid contact line on the two opposite sides ("uphill" or "downhill") of the drop. The required gradient in surface free energy was generated on the surface of a polished silicon wafer by exposing it to the diffusing front of a vapor of decyltrichlorosilane, Cl(3)Si(CH(2))(9)CH(3). The resulting surface displayed a gradient of hydrophobicity (with the contact angle of water changing from 97 degrees to 25 degrees ) over a distance of 1 centimeter. When the wafer was tilted from the horizontal plane by 15 degrees , with the hydrophobic end lower than the hydrophilic, and a drop of water (1 to 2 microliters) was placed at the hydrophobic end, the drop moved toward the hydrophilic end with an average velocity of approximately 1 to 2 millimeters per second. In order for the drop to move, the hysteresis in contact angle on the surface had to be low (相似文献   

Human symbionts use a host-like pathway for surface fucosylation

The mammalian intestine harbors a beneficial microbiota numbering approximately 10(12) organisms per gram of colonic content. The host tolerates this tremendous bacterial load while maintaining the ability to efficiently respond to pathogenic organisms. In this study, we show that the Bacteroides use a mammalian-like pathway to decorate numerous surface capsular polysaccharides and glycoproteins with l-fucose, an abundant surface molecule of intestinal epithelial cells, resulting in the coordinated expression of this surface molecule by host and symbiont. A Bacteroides mutant deficient in the ability to cover its surface with L-fucose is defective in colonizing the mammalian intestine under competitive conditions.     

Immunodeficiency and clonal growth of target cells induced by helper-free defective retrovirus

The murine acquired immunodeficiency syndrome is induced by a defective retrovirus. To study the role of virus replication in this disease, helper-free stocks of defective Duplan virus were produced. These stocks were highly pathogenic in absence of detectable replicating murine leukemia viruses (MuLVs) other than xenotropic MuLV. They induced expansion of the infected cell population (over 1000-fold), and this cell expansion was oligoclonal in origin and, most likely, arose through cell division. These results suggest that this defective virus is oncogenic, inducing a primary neoplasia associated with an acquired immunodeficiency syndrome as a paraneoplastic syndrome. These data emphasize the need to determine whether virus replication is necessary for the progression of other immunodeficiency diseases, including acquired immunodeficiency syndrome, and whether these diseases also represent paraneoplastic syndromes.     

Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes

In 2001, dengue virus type 1 (DENV-1) populations in humans and mosquitoes from Myanmar acquired a stop-codon mutation in the surface envelope (E) protein gene. Within a year, this stop-codon strain had spread to all individuals sampled. The presence of truncated E protein species within individual viral populations, along with a general relaxation in selective constraint, indicated that the stop-codon strain represents a defective lineage of DENV-1. We propose that such long-term transmission of defective RNA viruses in nature was achieved through complementation by coinfection of host cells with functional viruses.     

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.