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1.
Stefan Gerhardy Anna Maria Menet Cohue Peña Janusz Jurand Petkowski Vikram Govind Panse 《Chromosoma》2014,123(4):327-344
The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. In this review, we summarize our current understanding of nuclear export of pre-ribosomal subunits and cytoplasmic maturation steps that render pre-ribosomal subunits translation-competent. 相似文献
2.
Jomaa A Stewart G Martín-Benito J Zielke R Campbell TL Maddock JR Brown ED Ortega J 《RNA (New York, N.Y.)》2011,17(4):697-709
Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo. 相似文献
3.
Williamson JR 《Quarterly reviews of biophysics》2005,38(4):397-403
The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway. 相似文献
4.
Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly. 相似文献
5.
Jakovljevic J de Mayolo PA Miles TD Nguyen TM Léger-Silvestre I Gas N Woolford JL 《Molecular cell》2004,14(3):331-342
Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA. 相似文献
6.
Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are likely regulatory proteins, much work to date has focused on understanding their role in the assembly process. Here, we review these factors, as well as other sources of energy, and their roles in the ribosome assembly process. In addition, we propose roles of energy-releasing enzymes in the assembly process, to explain why energy is used for a process that occurs largely spontaneously in bacteria. Finally, we use literature data to suggest testable models for how these enzymes could be used as targets for regulation of ribosome assembly. 相似文献
7.
Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit 总被引:1,自引:0,他引:1
Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly. 相似文献
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9.
Regulation of ribosome biogenesis within the nucleolus. 总被引:1,自引:0,他引:1
10.
A three-fluorophore FRET assay for high-throughput screening of small-molecule inhibitors of ribosome assembly 总被引:1,自引:0,他引:1
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In one of the first steps of prokaryotic ribosome assembly, the ribosomal protein S15 binds to a three-way junction in the central domain of the 16S rRNA. Binding causes a conformational change that is required for subsequent binding events. Using a novel fluorescence resonance energy transfer assay with three fluorophores, two on the RNA and one on the S15 protein, small-molecule libraries can be screened for potential inhibitors of this initial step in ribosome assembly. The employment of three fluorophores allows both the conformational change of the RNA and the binding of S15 to be monitored in a single assay. 相似文献
11.
Jakob S Ohmayer U Neueder A Hierlmeier T Perez-Fernandez J Hochmuth E Deutzmann R Griesenbeck J Tschochner H Milkereit P 《PloS one》2012,7(3):e32552
Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains. 相似文献
12.
Elena Lavdovskaia Krt Denks Franziska Nadler Emely Steube Andreas Linden Henning Urlaub Marina V Rodnina Ricarda Richter-Dennerlein 《Nucleic acids research》2020,48(22):12929
Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria. 相似文献
13.
In Escherichia coli, rRNAs are initially transcribed with precursor sequences, which are subsequently removed through processing reactions. To investigate the role of precursor sequences, we analyzed ribosome assembly in strains containing mutations in the processing RNases. We observed that defects in 23S rRNA processing resulted in an accumulation of ribosomal subunits and caused a significant delay in ribosome assembly. These observations suggest that precursor residues in 23S rRNA control ribosome assembly and could be serving a regulatory role to couple ribosome assembly to rRNA processing. The possible mechanisms through which rRNA processing and ribosome assembly could be linked are discussed. 相似文献
14.
The motions of large systems such as the ribosome are not fully accessible with conventional molecular simulations. A coarse-grained, less-than-atomic-detail model such as the anisotropic network model (ANM) is a convenient informative tool to study the cooperative motions of the ribosome. The motions of the small 30S subunit, the larger 50S subunit, and the entire 70S assembly of the two subunits have been analyzed using ANM. The lowest frequency collective modes predicted by ANM show that the 50S subunit and 30S subunit are strongly anti-correlated in the motion of the 70S assembly. A ratchet-like motion is observed that corresponds well to the experimentally reported ratchet motion. Other slow modes are also examined because of their potential links to the translocation steps in the ribosome. We identify several modes that may facilitate the E-tRNA exiting from the assembly. The A-site t-RNA and P-site t-RNA are found to be strongly coupled and positively correlated in these slow modes, suggesting that the translocations of these two t-RNAs occur simultaneously, while the motions of the E-site t-RNA are less correlated, and thus less likely to occur simultaneously. Overall the t-RNAs exhibit relatively large deformations. Animations of these slow modes of motion can be viewed at. 相似文献
15.
The nucleolus is the site of ribosomal RNA synthesis, processing and ribosome maturation. Various small ribonucleoproteins also undergo maturation in the nucleolus, involving RNA modification and RNA-protein assembly. Such steps and other activities of small ribonucleoproteins also take place in Cajal (coiled) bodies. Events of ribosome biogenesis are found solely in the nucleolus, which is the final destination of small nucleolar RNAs after their traffic through Cajal bodies. However, nucleoli are just a stopping point in the intricate cellular traffic for small nuclear RNAs and other ribonucleoproteins. 相似文献
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Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5' domain of the Escherichia coli 16S rRNA at 20 ms intervals using X-ray-dependent hydroxyl radical footprinting. Comparison with crystallographic structures and footprinting reactions on native 30S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20 mM MgCl2, many tertiary interactions appeared within 20 ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly. 相似文献
19.
Treatment of the intact bacterial ribosome with proteolytic enzymes results in little or no digestion of many of the component proteins [Craven, G. R., & Gupta, V. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 1329]. In contrast, when the proteins are released from the constraints of ribosome structure they become completely susceptible to proteolytic attack. We have attempted to exploit these observations in an effort to determine the precise steps in ribosome assembly which result in a conversion of the structures of the various proteins from a proteolysis sensitive to a resistant form. Thus, a total of 11 30S ribosome assembly intermediate complexes of proteins and 16S RNA were prepared and digested with trypsin or chymotrypsin. The kinetics of digestion of each protein in the complex were followed by polyacrylamide gel electrophoresis. By a comparison of the digestion pattern of two complexes differing only by the presence of a single protein, it was possible to deduce several specific protective effects of one protein on its neighbor in the complex. On the basis of these studies, we propose nine protein-protein protective effects. The possible relevance of these interrelationships to other well-established proximity relationships is discussed. 相似文献
20.
Jonathan R. Warner 《EMBO reports》2010,11(1):6-8
The EMBO Conference on Ribosome Synthesis held last summer explored the latest breakthroughs in ribosome assembly and how it affects disease. Both of these topics have recently seen important advances that enlighten how almost 200 proteins cooperate to produce a ribosome and how the cell responds to a malfunction in this process.Medieval Regensburg, Germany played host from 26 to 30 August 2009 to more than 230 participants in the eighth triennial international meeting on ‘Ribosome Synthesis,” organized by Herbert Tschochner, Gernot Längst, Philipp Milkereit, David Tollervey and John Woolford. This meeting marked a clear step forward for two main reasons: a substantial advance in understanding the incredibly complex process of eukaryotic ribosome assembly, and an increasing appreciation of the fact that aberrant ribosome synthesis has a critical role in human diseases such as anaemia and cancer. 相似文献