Structure Of Ribosome Research Articles

Unusual biology across a group comprising more than 15% of domain Bacteria.

A prominent feature of the bacterial domain is a radiation of major lineages that are defined as candidate phyla because they lack isolated representatives. Bacteria from these phyla occur in diverse environments and are thought to mediate carbon and hydrogen cycles. Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation. Here we reconstructed 8 complete and 789 draft genomes from bacteria representing >35 phyla and documented features that consistently distinguish these organisms from other bacteria. We infer that this group, which may comprise >15% of the bacterial domain, has shared evolutionary history, and describe it as the candidate phyla radiation (CPR). All CPR genomes are small and most lack numerous biosynthetic pathways. Owing to divergent 16S ribosomal RNA (rRNA) gene sequences, 50-100% of organisms sampled from specific phyla would evade detection in typical cultivation-independent surveys. CPR organisms often have self-splicing introns and proteins encoded within their rRNA genes, a feature rarely reported in bacteria. Furthermore, they have unusual ribosome compositions. All are missing a ribosomal protein often absent in symbionts, and specific lineages are missing ribosomal proteins and biogenesis factors considered universal in bacteria. This implies different ribosome structures and biogenesis mechanisms, and underlines unusual biology across a large part of the bacterial domain.

Expression profiling of white sponge nevus by RNA sequencing revealed pathological pathways.

BackgroundWhite sponge nevus (WSN) is a rare periodontal hereditary disease. To date, almost all WSN studies have focused on case reports or mutation reports. Thus, the mechanism behind WSN is still unclear. We investigated the pathogenesis of WSN using expression profiling.MethodsSequence analysis of samples from a WSN Chinese family revealed a mutation (332 T > C) in the KRT13 gene that resulted in the amino acid change Leu111Pro. The pathological pathway behind the WSN expression profile was investigated by RNA sequencing (RNA-seq).ResultsConstruction of a heatmap revealed 24 activated genes and 57 reduced genes in the WSN patients. The ribosome structure was damaged in the WSN patients. Moreover, the translation rate was limited in the WSN patients, whereas ubiquitin-mediated proteolysis was enhanced.ConclusionsOur results suggest that the abnormal degradation of the KRT13 protein in WSN patients may be associated with keratin 7 (KRT7) and an abnormal ubiquitination process.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0285-y) contains supplementary material, which is available to authorized users.

Structural insights into the translational infidelity mechanism.

The decoding of mRNA on the ribosome is the least accurate process during genetic information transfer. Here we propose a unified decoding mechanism based on 11 high-resolution X-ray structures of the 70S ribosome that explains the occurrence of missense errors during translation. We determined ribosome structures in rare states where incorrect tRNAs were incorporated into the peptidyl-tRNA-binding site. These structures show that in the codon–anticodon duplex, a G·U mismatch adopts the Watson–Crick geometry, indicating a shift in the tautomeric equilibrium or ionization of the nucleobase. Additional structures with mismatches in the 70S decoding centre show that the binding of any tRNA induces identical rearrangements in the centre, which favours either isosteric or close to the Watson–Crick geometry codon–anticodon pairs. Overall, the results suggest that a mismatch escapes discrimination by preserving the shape of a Watson–Crick pair and indicate that geometric selection via tautomerism or ionization dominates the translational infidelity mechanism.

About the ribosomal biogenesis in human

Ribosomes are cellular ribonucleoprotein particles required for a fundamental mechanism, translation of the genetic information into proteins. Ribosome biogenesis is a highly complex pathway involving many maturation steps: ribosomal RNA (rRNA) synthesis, rRNA processing, pre-rRNA modifications, its assembly with ribosomal proteins in the nuceolus, export of the subunit precursors to the nucleoplasm and the cytoplasm. Ribosome biogenesis has mainly being investigated in yeast during these last 25 years. However, recent works have shown that, despite many similarities between yeast and human ribosome structure and biogenesis, human pre-rRNA processing is far more complex than in yeast. In order to better understand diseases related to a malfunction in ribosome synthesis, the ribosomopathies, research should be conducted directly in human cells and animal models.

Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A

The increase in multi-drug-resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P-, and E-site tRNAs bound and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

69 Quantifying the energy landscapes of ribosome function through simulation

The breadth of information available on ribosome structure and dynamics makes it the ideal system for systematically investigating the physical-chemical properties that enable large-scale biologica...

Structure of the Bacillus subtilis 70S ribosome reveals the basis for species-specific stalling.

Ribosomal stalling is used to regulate gene expression and can occur in a species-specific manner. Stalling during translation of the MifM leader peptide regulates expression of the downstream membrane protein biogenesis factor YidC2 (YqjG) in Bacillus subtilis, but not in Escherichia coli. In the absence of structures of Gram-positive bacterial ribosomes, a molecular basis for species-specific stalling has remained unclear. Here we present the structure of a Gram-positive B. subtilis MifM-stalled 70S ribosome at 3.5–3.9 Å, revealing a network of interactions between MifM and the ribosomal tunnel, which stabilize a non-productive conformation of the PTC that prevents aminoacyl-tRNA accommodation and thereby induces translational arrest. Complementary genetic analyses identify a single amino acid within ribosomal protein L22 that dictates the species specificity of the stalling event. Such insights expand our understanding of how the synergism between the ribosome and the nascent chain is utilized to modulate the translatome in a species-specific manner.

Structure of the human 80S ribosome.

Ribosomes are translational machineries that catalyse protein synthesis. Ribosome structures from various species are known at the atomic level, but obtaining the structure of the human ribosome has remained a challenge; efforts to address this would be highly relevant with regard to human diseases. Here we report the near-atomic structure of the human ribosome derived from high-resolution single-particle cryo-electron microscopy and atomic model building. The structure has an average resolution of 3.6Å, reaching 2.9Å resolution in the most stable regions. It provides unprecedented insights into ribosomal RNA entities and amino acid side chains, notably of the transfer RNA binding sites and specific molecular interactions with the exit site tRNA. It reveals atomic details of the subunit interface, which is seen to remodel strongly upon rotational movements of the ribosomal subunits. Furthermore, the structure paves the way for analysing antibiotic side effects and diseases associated with deregulated protein synthesis.

Structural insights into ribosome structure and translation regulation

Protein synthesis (translation) is accomplished by ribosomes in all living cells. Ribosomes decode genetic information encoded in messenger RNA and catalyze peptide‐bond formation. We study the mechanisms of translation translation regulation, using structural and biochemical methods. Here, we will present new structural insights into regulation of translation on the ribosome under cellular stress and/or disease.

A conserved transcriptional signature of delayed aging and reduced disease vulnerability is partially mediated by SIRT3.

Aging is the most significant risk factor for a range of diseases, including many cancers, neurodegeneration, cardiovascular disease, and diabetes. Caloric restriction (CR) without malnutrition delays aging in diverse species, and therefore offers unique insights into age-related disease vulnerability. Previous studies suggest that there are shared mechanisms of disease resistance associated with delayed aging, however quantitative support is lacking. We therefore sought to identify a common response to CR in diverse tissues and species and determine whether this signature would reflect health status independent of aging. We analyzed gene expression datasets from eight tissues of mice subjected to CR and identified a common transcriptional signature that includes functional categories of mitochondrial energy metabolism, inflammation and ribosomal structure. This signature is detected in flies, rats, and rhesus monkeys on CR, indicating aspects of CR that are evolutionarily conserved. Detection of the signature in mouse genetic models of slowed aging indicates that it is not unique to CR but rather a common aspect of extended longevity. Mice lacking the NAD-dependent deacetylase SIRT3 fail to induce mitochondrial and anti-inflammatory elements of the signature in response to CR, suggesting a potential mechanism involving SIRT3. The inverse of this transcriptional signature is detected with consumption of a high fat diet, obesity and metabolic disease, and is reversed in response to interventions that decrease disease risk. We propose that this evolutionarily conserved, tissue-independent, transcriptional signature of delayed aging and reduced disease vulnerability is a promising target for developing therapies for age-related diseases.

Back to the future of RNA structure.

Back to the future of RNA structure.

Fifty to 100 times the size!

In recollecting on discoveries in the RNA field over the last 20 years, one can find of course many, considering the phenomenal field we are all working in. But of course as a structural biologist, I remember vividly the day I saw for the first time a talk presenting the high resolution structure of the bacterial 50S ribosomal particle. If I do recollect well, the 10-minute talk was presented by Poul Nissen (from Tom Steitz's group in Yale) as the opening talk of the 2000 RNA Society meeting in Madison. I was just stunned and felt almost oppressed by what was shown in front of my eyes. The structure of this 1.5 megadalton protein–RNA complex containing 1500 nucleotides solved at 2.4 A, was 16 times larger than the largest RNA solved to date (the P4-P6 group I intron from the Doudna lab) and more than 50 times larger than the protein–RNA complex I was personally working on at this time (Nucleolin tandem RRMs bound to RNA that I did as a postdoc in the Feigon lab). Suddenly, I and probably many others present in the auditorium were of course in awe of such an accomplishment and of the beauty of this large particle, and at the same time probably felt insignificant when comparing this achievement with our own. This compares with the participants of the 100 m dash at the 2008 Olympic games in Beijing who were running behind Usain Bolt and saw him winning with such a margin that today probably nobody remembers who finished second with the silver medal. I still remember how silent the Madison auditorium was at the end of the presentation and I am sure that few remember what was presented afterwards in this session. This was so astonishing. I gave a talk on the nucleolin structure in the same session but fortunately after the coffee break. I am not sure that I would have been able to give this talk if I had been in the first half! Of course, later people only talked about the ribosome structure during the meeting and several structural biologists and ribosome experts thought that they were now “out of business.” In reality, this structure together with the one of the 30S ribosome particle by the Ramakrishnan and Yonath labs revitalized the ribosome field, a field still today very active and competitive as evidenced by the numerous structures of bacterial, eukaryotic, and mitochondrial ribosomes that were published at high resolution by the labs of Noller, Cate, Ban, and the three labs mentioned earlier that shared the Nobel prize for chemistry in 2009. This was 15 years ago and most of us are fortunately not “out of business.” The ribosome in the end is probably the exception rather than the norm and solving the structure of an RNP still remains a challenging project for NMR, crystallography, or electron microscopy even if the complex is 100 times smaller than the bacterial 50S particle or the eukaryotic ribosome. The structural biology questions ahead of us in the RNP field do not concern necessarily large particles but rather for example how RNPs forming on messenger RNA dynamically change at the different stages of the life of an mRNA. To understand this, small RNA binding domains and their attached disordered tails and medium size RNPs like the UsnRNPs will still need to be characterized structurally. Moreover, we will need to know exactly where and when they bind and dissociate from the mRNA. We are still not there yet although nobody knows if a new revolution similar to the one we saw in 2000 is not waiting to blow our minds again.

An origin story: ribozyme catalysis by the ribosome.

An origin story: ribozyme catalysis by the ribosome.

Translational control 1995-2015: unveiling molecular underpinnings and roles in human biology.

The last 20 years have seen tremendous progress in deciphering the fundamental mechanisms of protein synthesis and molecular pathways of translational control, and in implicating these processes in development, neurobiology, and etiologyof human diseases. Muchof this success can be attributed to the introduction of new technologies, such as next-generation RNA sequencing and advances in cryo-EM; and also to development of powerful in vitro systems recapitulating complex in vivo phenomena with purified components. Also critical have been strategic interactions between experts in different disciplines of biology, such as the wedding of biochemistry with genetics and structural biology, and of translation biochemistry with neurobiology and medicine. RNA has contributed to this progress by publishing key findings on molecular mechanisms of protein synthesis and the role of translational control in regulating gene expression in diverse organisms. Thebasic reactions of the elongationand termination stages of protein synthesis are highly conserved throughout all three kingdoms of life. Because they occur in the catalytic center of the ribosome, their molecular mechanisms were illuminated by the stunning successes of X-ray crystallography and cryo-electron microscopy in producing high-resolution structures of both empty ribosomes and a myriad of ribosome complexes with mRNA, tRNAs, or elongation, termination, or ribosome recycling factors trapped in different stages of the process. Recent innovations in cryo-EM technology are revealing ribosome structures at nearly 3 A resolution; and new algorithms for sorting structurally distinct molecules means that sample heterogeneity (an anathema to crystallography) is now exploited to identify different conformational states in the same preparation. The solved ribosome structures revealed the path of mRNA and the A, P, and E binding sites for the tRNA, as it progress through the elongation cycle from aminoacyltRNA, to peptidyl-tRNA to deacylated tRNA, located at the interface between the large and small subunits; as well as the peptidyl transferase center wherepeptide bond formation occurs. As the decoding center of the ribosome is comprised almost entirely of rRNA, it became clear that the ribosome is a ribozyme. The protein factors involved in translocation of mRNA and the tRNAs through the decoding sites (EF-G/ EF2), in recognizing stop codons and hydrolyzing peptidyltRNA (RF1/eRF1), and splitting apart ribosomal subunits following termination (bacterial RRF) all exploit striking structural mimicry of tRNA to fit into the A site. Interestingly, RF1 was shown to contain a protein element that “reads” the stop codon and another that reaches into the peptidyl transferase center to hydrolyze the peptidyl-tRNA linkage. The peptidyl-tRNA and aminoacyl-tRNA are anchored in the precise locations required for peptide bond formation, but the ribosome is not simply a rigid catalytic platform, but undergoes conformational changes in selecting the correct EF1A·GTP·aminoacyl-tRNA ternary complex for each codon, and displays inter-subunit ratcheting during translocation and termination. GTP binding and hydrolysis by the elongation factors evoke cyclic conformational changes that drive the process forward. The use of fluorescently labeled reactants and stopped-flow kinetic analysis allowed a detailed dissection of different steps in the processes of aminoacyltRNA selection in the A site, with proofreading of the codon:anticodon interaction; and of translocation of tRNAs through the decoding center catalyzed by EF2. Single-molecule approaches also contributed importantly to this progress, by identifying intermediate states too transient to be detected by ensemble kinetic experiments or structural biology. In other developments, eIF5A and its bacterial counterpart EF-P were shown to stimulate elongation specifically through stretches of proline codons, and both factors occupy the E-site and communicate directly with the peptidyl transferase center. Interestingly, RRF is replaced in eukaryotes and archaea by an ATP-binding cassette protein, ABCE1/ Rli1, that becomes wedged between the large and small subunits and uses ATP hydrolysis to split apart the subunits. ABCE1/Rli1also functions with RF1 paralog Dom34 to

Twenty years of RNA crystallography.

Twenty years of RNA crystallography.

Ribosome biochemistry in crystal structure determination.

Protein synthesis is carried out by ribosomes, the universal ribonucleoprotein assemblies, where genetic information encoded in messenger RNA is translated into a polypeptide chain. These giant organelles, which have a molecular weight of ∼2.5 MDa in bacteria and up to 4.5 MDa in higher organisms, are composed of many different proteins and long RNA chains. Although the ribosome and its basic functions were discovered more than 50 years ago, the large size (approximately 4600 nucleotides and 55 proteins) and highly dynamic nature of this macromolecule made it difficult to obtain ribosomes in crystal form. The first X-ray structures of whole bacterial ribosome, 30S bacterial, and 50S archaea subunits triggered an avalanche of new works in the ribosome field, which vastly increased our understanding of the functioning of this molecular machine.

The early days of RNomics.

In the late eighties, I became interested in non-protein-coding RNAs (ncRNAs) during the completion of my PhD in Munich (Germany) and continued my interest, as a postdoc, in one of the main centers for ribosomal RNA and protein synthesis research, the lab of Harry Noller at UCSC in California (USA). Shortly after I arrived at Harry's lab, he published one of his hallmark papers in Science, demonstrating that ribosomal RNA, mainly devoid of ribosomal proteins, was able to catalyze protein synthesis. This finding implied a catalytic function for ribosomal RNA rather than a scaffolding function. Many of his former students and postdocs, me included, still regret that Harry didn't receive the Nobel prize for his pioneering work on ribosomes. After all, his lab was the first to sequence the 16S and 23S RNAs (rRNAs) from E. coli, proposed the first 2D and 3D structure models of rRNAs and the ribosome, and also investigated, by chemical probing, its interaction with tRNA and antibiotics. In addition, he was the first to publish the crystal structure of the 70S ribosome. I know many scientists who would not have taken it lightly, when they missed the Nobel prize by such a small margin, as Harry probably did. However, when I once asked Harry whether he would like to receive the Nobel Prize at some point in his career, he answered: “Hell no, I would rather know how the ribosome really works…”.

Splicing: still so much to learn.

Splicing: still so much to learn.

Reflections on 20 years of RNA.

Reflections on 20 years of RNA.

Twenty years of ribosome assembly and ribosomopathies.

During the 20 years of RNA extraordinary progress has been made in the study of ribosomes, beyond that of ribosome structure, which will be well covered by others.