Journal articles: 'Ribosome binding sites'

1

Márquez, V., D. N. Wilson, and K. H. Nierhaus. "Functions and interplay of the tRNA-binding sites of the ribosome." Biochemical Society Transactions 30, no. 2 (April 1, 2002): 133–40. http://dx.doi.org/10.1042/bst0300133.

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The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids. The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e. an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins. Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites. In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals. These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA · EF-Tu · GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA2 · mRNA complex during translocation and the maintenance of the reading frame.

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Dorner, S., and A. Barta. "Probing Ribosome Structure by Europium-Induced RNA Cleavage." Biological Chemistry 380, no. 2 (February 1, 1999): 243–51. http://dx.doi.org/10.1515/bc.1999.032.

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AbstractDivalent metal ions are absolutely required for the structure and catalytic activities of ribosomes. They are partly coordinated to highly structured RNA, which therefore possesses high-affinity metal ion binding pockets. As metalion induced RNA cleavages are useful for characterising metal ion binding sites and RNA structures, we analysed europium (Eu3+) induced specific cleavages in both 16S and 23S rRNA ofE. coli. The cleavage sites were identified by primer extension and compared to those previously identified for calcium, lead, magnesium, and manganese ions. Several Eu3+cleavage sites, mostly those at which a general metal ion binding site had been already identified, were identical to previously described divalent metal ions. Overall, the Eu3+cleavages are most similar to the Ca2+cleavage pattern, probably due to a similar ion radius. Interestingly, several cleavage sites which were specific for Eu3+were located in regions implicated in the binding of tRNA and antibiotics. The binding of erythromycin and chloramphenicol, but not tetracycline and streptomycin, significantly reduced Eu3+cleavage efficiencies in the peptidyl transferase center. The identification of specific Eu3+binding sites near the active sites on the ribosome will allow to use the fluorescent properties of europium for probing the environment of metal ion binding pockets at the ribosome's active center.

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Prinz, Anke, Enno Hartmann, and Kai-Uwe Kalies. "Sec61p Is the Main Ribosome Receptor in the Endoplasmic Reticulum of Saccharomyces cerevisiae." Biological Chemistry 381, no. 9-10 (September 13, 2000): 1025–28. http://dx.doi.org/10.1515/bc.2000.126.

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Abstract A characteristic feature of the co-translational protein translocation into the endoplasmic reticulum (ER) is the tight association of the translating ribosomes with the translocation sites in the membrane. Biochemical analyses identified the Sec61 complex as the main ribosome receptor in the ER of mammalian cells. Similar experiments using purified homologues from the yeast Saccharomyces cerevisiae, the Sec61p complex and the Ssh1p complex, respectively, demonstrated that they bind ribosomes with an affinity similar to that of the mammalian Sec61 complex. However, these studies did not exclude the presence of other proteins that may form abundant ribosome binding sites in the yeast ER. We now show here that similar to the situation found in mammals in the yeast Saccharomyces cerevisiae the two Sec61-homologues Sec61p and Ssh1p are essential for the formation of high-affinity ribosome binding sites in the ER membrane. The number of binding sites formed by Ssh1p under standard growth conditions is at least 4 times less than those formed by Sec61p.

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4

Schaletzky, Julia, and Tom A. Rapoport. "Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane." Molecular Biology of the Cell 17, no. 9 (September 2006): 3860–69. http://dx.doi.org/10.1091/mbc.e06-05-0439.

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We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC–SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC–SRP complexes can overcome the competition by nontranslating ribosomes.

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Krüger, Tim, Hanswalter Zentgraf, and Ulrich Scheer. "Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins." Journal of Cell Biology 177, no. 4 (May 21, 2007): 573–78. http://dx.doi.org/10.1083/jcb.200612048.

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Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC–GC interface.

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Auerbach, Tamar, Inbal Mermershtain, Chen Davidovich, Anat Bashan, Matthew Belousoff, Itai Wekselman, Ella Zimmerman, et al. "The structure of ribosome-lankacidin complex reveals ribosomal sites for synergistic antibiotics." Proceedings of the National Academy of Sciences 107, no. 5 (January 11, 2010): 1983–88. http://dx.doi.org/10.1073/pnas.0914100107.

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Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.

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Lytle, J. Robin, Lily Wu, and Hugh D. Robertson. "The Ribosome Binding Site of Hepatitis C Virus mRNA." Journal of Virology 75, no. 16 (August 15, 2001): 7629–36. http://dx.doi.org/10.1128/jvi.75.16.7629-7636.2001.

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ABSTRACT Hepatitis C virus (HCV) infects an estimated 170 million people worldwide, the majority of whom develop a chronic infection which can lead to severe liver disease, and for which no generally effective treatment yet exists. A promising target for treatment is the internal ribosome entry site (IRES) of HCV, a highly conserved domain within a highly variable RNA. Never before have the ribosome binding sites of any IRES domains, cellular or viral, been directly characterized. Here, we reveal that the HCV IRES sequences most closely associated with 80S ribosomes during protein synthesis initiation are a series of discontinuous domains together comprising by far the largest ribosome binding site yet discovered.

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Wittmann-Liebold, Brigitte, Monika Ühlein, Henning Urlaub, Eva-Christina Müller, Albrecht Otto, and Oliver Bischof. "Structural and functional implications in the eubacterial ribosome as revealed by protein–rRNA and antibiotic contact sites." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1187–97. http://dx.doi.org/10.1139/o95-128.

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Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2–iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, andL36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at die peptide level in several proteins that were found to constitute me antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, and L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA–peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 widi a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.Key words: antibiotic-binding site, RNA–peptide-binding sites, protein–RNA interaction in ribosomes, functional role of protein L2.

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9

Espah Borujeni, Amin, Anirudh S. Channarasappa, and Howard M. Salis. "Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites." Nucleic Acids Research 42, no. 4 (November 14, 2013): 2646–59. http://dx.doi.org/10.1093/nar/gkt1139.

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Abstract The ribosome’s interactions with mRNA govern its translation rate and the effects of post-transcriptional regulation. Long, structured 5′ untranslated regions (5′ UTRs) are commonly found in bacterial mRNAs, though the physical mechanisms that determine how the ribosome binds these upstream regions remain poorly defined. Here, we systematically investigate the ribosome’s interactions with structured standby sites, upstream of Shine–Dalgarno sequences, and show that these interactions can modulate translation initiation rates by over 100-fold. We find that an mRNA’s translation initiation rate is controlled by the amount of single-stranded surface area, the partial unfolding of RNA structures to minimize the ribosome’s binding free energy penalty, the absence of cooperative binding and the potential for ribosomal sliding. We develop a biophysical model employing thermodynamic first principles and a four-parameter free energy model to accurately predict the ribosome’s translation initiation rates for 136 synthetic 5′ UTRs with large structures, diverse shapes and multiple standby site modules. The model predicts and experiments confirm that the ribosome can readily bind distant standby site modules that support high translation rates, providing a physical mechanism for observed context effects and long-range post-transcriptional regulation.

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10

Kalies, K. U., D. Görlich, and T. A. Rapoport. "Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex." Journal of Cell Biology 126, no. 4 (August 15, 1994): 925–34. http://dx.doi.org/10.1083/jcb.126.4.925.

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The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.

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11

Dantley, Kathi A., H. Kathleen Dannelly, and Vickers Burdett. "Binding Interaction between Tet(M) and the Ribosome: Requirements for Binding." Journal of Bacteriology 180, no. 16 (August 15, 1998): 4089–92. http://dx.doi.org/10.1128/jb.180.16.4089-4092.1998.

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ABSTRACT Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5′-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.

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12

Barrick, Doug, Keith Villanueba, John Childs, Rhonda Kalil, Thomas D. Schneider, Charles E. Lawrence, Larry Gold, and Gary D. Stormo. "Quantitative analysis of ribosome binding sites in E.coli." Nucleic Acids Research 22, no. 7 (1994): 1287–95. http://dx.doi.org/10.1093/nar/22.7.1287.

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13

Raden, David, Weiqun Song, and Reid Gilmore. "Role of the Cytoplasmic Segments of Sec61α in the Ribosome-Binding and Translocation-Promoting Activities of the Sec61 Complex." Journal of Cell Biology 150, no. 1 (July 10, 2000): 53–64. http://dx.doi.org/10.1083/jcb.150.1.53.

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The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH2 and COOH terminus of Sec61α was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61α. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.

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14

Yan, Kang, Eric Hunt, John Berge, Earl May, Robert A. Copeland, and Richard R. Gontarek. "Fluorescence Polarization Method To Characterize Macrolide-Ribosome Interactions." Antimicrobial Agents and Chemotherapy 49, no. 8 (August 2005): 3367–72. http://dx.doi.org/10.1128/aac.49.8.3367-3372.2005.

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ABSTRACT A fluorescence polarization assay is described that measures the binding of fluorescently labeled erythromycin to 70S ribosomes from Escherichia coli and the displacement of the erythromycin from these ribosomes. The assay has been validated with several macrolide derivatives and other known antibiotics. We demonstrate that this assay is suitable for determining the dissociation constants of novel compounds that have binding sites overlapping those of macrolides. This homogeneous binding assay provides a valuable tool for defining structure-activity relationships among compounds during the discovery and development of new ribosome-targeting drugs.

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Ehrenbolger, Kai, Nathan Jespersen, Himanshu Sharma, Yuliya Y. Sokolova, Yuri S. Tokarev, Charles R. Vossbrinck, and Jonas Barandun. "Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome." PLOS Biology 18, no. 10 (October 30, 2020): e3000958. http://dx.doi.org/10.1371/journal.pbio.3000958.

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Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo–electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein–protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

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Caban, Kelvin, Scott A. Kinzy, and Paul R. Copeland. "The L7Ae RNA Binding Motif Is a Multifunctional Domain Required for the Ribosome-Dependent Sec Incorporation Activity of Sec Insertion Sequence Binding Protein 2." Molecular and Cellular Biology 27, no. 18 (July 16, 2007): 6350–60. http://dx.doi.org/10.1128/mcb.00632-07.

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ABSTRACT The decoding of specific UGA codons as selenocysteine is specified by the Sec insertion sequence (SECIS) element. Additionally, Sec-tRNA[Ser]Sec and the dedicated Sec-specific elongation factor eEFSec are required but not sufficient for nonsense suppression. SECIS binding protein 2 (SBP2) is also essential for Sec incorporation, but its precise role is unknown. In addition to binding the SECIS element, SBP2 binds stably and quantitatively to ribosomes. To determine the function of the SBP2-ribosome interaction, conserved amino acids throughout the SBP2 L7Ae RNA binding motif were mutated to alanine in clusters of five. Mutant proteins were analyzed for ribosome binding, SECIS element binding, and Sec incorporation activity, allowing us to identify two distinct but interdependent sites within the L7Ae motif: (i) a core L7Ae motif required for SECIS binding and ribosome binding and (ii) an auxiliary motif involved in physical and functional interactions with the ribosome. Structural modeling of SBP2 based on the 15.5-kDa protein-U4 snRNA complex strongly supports a two-site model for L7Ae domain function within SBP2. These results provide evidence that the SBP2-ribosome interaction is essential for Sec incorporation.

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Zhang, Ling, Ying-Hui Wang, Xing Zhang, Laura Lancaster, Jie Zhou, and Harry F. Noller. "The structural basis for inhibition of ribosomal translocation by viomycin." Proceedings of the National Academy of Sciences 117, no. 19 (April 27, 2020): 10271–77. http://dx.doi.org/10.1073/pnas.2002888117.

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Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

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18

Planta, Rudi J., Paula M. Gonçalves, and Willem H. Mager. "Global regulators of ribosome biosynthesis in yeast." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 825–34. http://dx.doi.org/10.1139/o95-090.

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Three abundant ubiquitous DNA-binding protein factors appear to play a major role in the control of ribosome biosynthesis in yeast. Two of these factors mediate the regulation of transcription of ribosomal protein genes (rp-genes) in yeasts. Most yeast rp-genes are under transcriptional control of Rap1p (repressor–activator protein), while a small subset of rp-genes is activated through Abf1p (ARS binding factor). The third protein, designated Reb1p (rRNA enhancer binding protein), which binds strongly to two sites located upstream of the enhancer and the promoter of the rRNA operon, respectively, appears to play a crucial role in the efficient transcription of the chromosomal rDNA. All three proteins, however, have many target sites on the yeast genome, in particular, in the upstream regions of several Pol II transcribed genes, suggesting that they play a much more general role than solely in the regulation of ribosome biosynthesis. Furthermore, some evidence has been obtained suggesting that these factors influence the chromatin structure and create a nucleosome-free region surrounding their binding sites. Recent studies indicate that the proteins can functionally replace each other in various cases and that they act synergistically with adjacent additional DNA sequences. These data suggest that Abf1p, Rap1p, and Reb1p are primary DNA-binding proteins that serve to render adjacent cis-acting elements accessible to specific trans-acting factors.Key words: Abf1p, Rap1p, Reb1p, yeast, ribosome biosynthesis.

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Ehrenberg, Måns, Nese Bilgin, Vildan Dincbas, Reza Karimi, Diarmaid Hughes, and Farhad Abdulkarim. "tRNA–ribosome interactions." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1049–54. http://dx.doi.org/10.1139/o95-112.

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Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-fRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I – domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I – domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation–diffusion techniques.Key words: ribosome, EF-Tu, accuracy, in vitro translation.

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Smethurst, Daniel G. J., Nikolay Kovalev, Erica R. McKenzie, Dimitri G. Pestov, and Natalia Shcherbik. "Iron-mediated degradation of ribosomes under oxidative stress is attenuated by manganese." Journal of Biological Chemistry 295, no. 50 (October 9, 2020): 17200–17214. http://dx.doi.org/10.1074/jbc.ra120.015025.

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Protein biosynthesis is fundamental to cellular life and requires the efficient functioning of the translational machinery. At the center of this machinery is the ribosome, a ribonucleoprotein complex that depends heavily on Mg2+ for structure. Recent work has indicated that other metal cations can substitute for Mg2+, raising questions about the role different metals may play in the maintenance of the ribosome under oxidative stress conditions. Here, we assess ribosomal integrity following oxidative stress both in vitro and in cells to elucidate details of the interactions between Fe2+ and the ribosome and identify Mn2+ as a factor capable of attenuating oxidant-induced Fe2+-mediated degradation of rRNA. We report that Fe2+ promotes degradation of all rRNA species of the yeast ribosome and that it is bound directly to RNA molecules. Furthermore, we demonstrate that Mn2+ competes with Fe2+ for rRNA-binding sites and that protection of ribosomes from Fe2+-mediated rRNA hydrolysis correlates with the restoration of cell viability. Our data, therefore, suggest a relationship between these two transition metals in controlling ribosome stability under oxidative stress.

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Unoson, Cecilia, and E. Gerhart H. Wagner. "Dealing with stable structures at ribosome binding sites: Bacterial translation and ribosome standby." RNA Biology 4, no. 3 (July 2007): 113–17. http://dx.doi.org/10.4161/rna.4.3.5350.

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Kimura, Takatsugu, Kuniaki Takagi, Yuya Hirata, Yoichi Hase, Akira Muto, and Hyouta Himeno. "Ribosome-Small-Subunit-Dependent GTPase Interacts with tRNA-Binding Sites on the Ribosome." Journal of Molecular Biology 381, no. 2 (August 2008): 467–77. http://dx.doi.org/10.1016/j.jmb.2008.06.023.

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23

Johnson, Alex G., Rosslyn Grosely, Alexey N. Petrov, and Joseph D. Puglisi. "Dynamics of IRES-mediated translation." Philosophical Transactions of the Royal Society B: Biological Sciences 372, no. 1716 (March 19, 2017): 20160177. http://dx.doi.org/10.1098/rstb.2016.0177.

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Viral internal ribosome entry sites (IRESs) are unique RNA elements, which use stable and dynamic RNA structures to recruit ribosomes and drive protein synthesis. IRESs overcome the high complexity of the canonical eukaryotic translation initiation pathway, often functioning with a limited set of eukaryotic initiation factors. The simplest types of IRESs are typified by the cricket paralysis virus intergenic region (CrPV IGR) and hepatitis C virus (HCV) IRESs, both of which independently form high-affinity complexes with the small (40S) ribosomal subunit and bypass the molecular processes of cap-binding and scanning. Owing to their simplicity and ribosomal affinity, the CrPV and HCV IRES have been important models for structural and functional studies of the eukaryotic ribosome during initiation, serving as excellent targets for recent technological breakthroughs in cryogenic electron microscopy (cryo-EM) and single-molecule analysis. High-resolution structural models of ribosome : IRES complexes, coupled with dynamics studies, have clarified decades of biochemical research and provided an outline of the conformational and compositional trajectory of the ribosome during initiation. Here we review recent progress in the study of HCV- and CrPV-type IRESs, highlighting important structural and dynamics insights and the synergy between cryo-EM and single-molecule studies. This article is part of the themed issue ‘Perspectives on the ribosome’.

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Au, Hilda H., Gabriel Cornilescu, Kathryn D. Mouzakis, Qian Ren, Jordan E. Burke, Seonghoon Lee, Samuel E. Butcher, and Eric Jan. "Global shape mimicry of tRNA within a viral internal ribosome entry site mediates translational reading frame selection." Proceedings of the National Academy of Sciences 112, no. 47 (November 9, 2015): E6446—E6455. http://dx.doi.org/10.1073/pnas.1512088112.

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The dicistrovirus intergenic region internal ribosome entry site (IRES) adopts a triple-pseudoknotted RNA structure and occupies the core ribosomal E, P, and A sites to directly recruit the ribosome and initiate translation at a non-AUG codon. A subset of dicistrovirus IRESs directs translation in the 0 and +1 frames to produce the viral structural proteins and a +1 overlapping open reading frame called ORFx, respectively. Here we show that specific mutations of two unpaired adenosines located at the core of the three-helical junction of the honey bee dicistrovirusIsraeli acute paralysis virus(IAPV) IRES PKI domain can uncouple 0 and +1 frame translation, suggesting that the structure adopts distinct conformations that contribute to 0 or +1 frame translation. Using a reconstituted translation system, we show that ribosomes assembled on mutant IRESs that direct exclusive 0 or +1 frame translation lack reading frame fidelity. Finally, a nuclear magnetic resonance/small-angle X-ray scattering hybrid approach reveals that the PKI domain of the IAPV IRES adopts an RNA structure that resembles a complete tRNA. The tRNA shape-mimicry enables the viral IRES to gain access to the ribosome tRNA-binding sites and form intermolecular contacts with the ribosome that are necessary for initiating IRES translation in a specific reading frame.

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David-Eden, Hilda, Alexander S. Mankin, and Yael Mandel-Gutfreund. "Structural signatures of antibiotic binding sites on the ribosome." Nucleic Acids Research 38, no. 18 (May 21, 2010): 5982–94. http://dx.doi.org/10.1093/nar/gkq411.

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May, E. E., M. A. Vouk, and D. L. Bitzer. "Classification of Escherichia coli K-12 ribosome binding sites." IEEE Engineering in Medicine and Biology Magazine 25, no. 1 (January 2006): 90–97. http://dx.doi.org/10.1109/memb.2006.1578668.

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Buddeweg, Anne, Kundan Sharma, Henning Urlaub, and Ruth A. Schmitz. "sRNA41affects ribosome binding sites within polycistronic mRNAs inMethanosarcina mazeiGö1." Molecular Microbiology 107, no. 5 (January 18, 2018): 595–609. http://dx.doi.org/10.1111/mmi.13900.

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Prokhorova, Irina, Roger B. Altman, Muminjon Djumagulov, Jaya P. Shrestha, Alexandre Urzhumtsev, Angelica Ferguson, Cheng-Wei Tom Chang, Marat Yusupov, Scott C. Blanchard, and Gulnara Yusupova. "Aminoglycoside interactions and impacts on the eukaryotic ribosome." Proceedings of the National Academy of Sciences 114, no. 51 (December 5, 2017): E10899—E10908. http://dx.doi.org/10.1073/pnas.1715501114.

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Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6′-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.

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Tate, Warren P., Elizabeth S. Poole, Julie A. Horsfield, Sally A. Mannering, Chris M. Brown, John G. Moffat, Mark E. Dalphin, Kim K. McCaughan, Louise L. Major, and Daniel N. Wilson. "Translational termination efficiency in both bacteria and mammals is regulated by the base following the stop codon." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1095–103. http://dx.doi.org/10.1139/o95-118.

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The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl–tRNA hydrolysis activity and enhanced codon–ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where −1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol −1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.Key words: translational stop signal, decoding, release factor, frame-shifting.

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Huang, Chih-Ting, Yei-Chen Lai, Szu-Yun Chen, Meng-Ru Ho, Yun-Wei Chiang, and Shang-Te Danny Hsu. "Structural polymorphism and substrate promiscuity of a ribosome-associated molecular chaperone." Magnetic Resonance 2, no. 1 (June 4, 2021): 375–86. http://dx.doi.org/10.5194/mr-2-375-2021.

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Abstract. Trigger factor (TF) is a highly conserved multi-domain molecular chaperone that exerts its chaperone activity at the ribosomal tunnel exit from which newly synthesized nascent chains emerge. TF also displays promiscuous substrate binding for a large number of cytosolic proteins independent of ribosome binding. We asked how TF recognizes a variety of substrates while existing in a monomer–dimer equilibrium. Paramagnetic nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy were used to show that dimeric TF displays a high degree of structural polymorphism in solution. A series of peptides has been generated to quantify their TF binding affinities in relation with their sequence compositions. The results confirmed a previous predication that TF preferentially binds to peptide fragments that are rich in aromatic and positively charged amino acids. NMR paramagnetic relaxation enhancement analysis showed that TF utilizes multiple binding sites, located in the chaperone domain and part of the prolyl trans–cis isomerization domain, to interact with these peptides. Dimerization of TF effectively sequesters most of the substrate binding sites, which are expected to become accessible upon binding to the ribosome as a monomer. As TF lacks ATPase activity, which is commonly used to trigger conformational changes within molecular chaperones in action, the ribosome-binding-associated disassembly and conformational rearrangements may be the underlying regulatory mechanism of its chaperone activity.

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NEKHAI, Sergei A., Vladimir E. BELETZKIJ, and Dmitri M. GRAIFER. "Influence of systematic error on the shape of the Scatchard plot of tRNAPhe binding to eukaryotic ribosomes." Biochemical Journal 325, no. 2 (July 15, 1997): 401–4. http://dx.doi.org/10.1042/bj3250401.

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Scatchard plots of tRNAPhe binding to poly(U)-programmed human 80 S ribosomes can be curved, either concave upwards or concave downwards, depending on the experimental conditions. The influence of a systematic error on the shape of the Scatchard plots has been analysed in a model experiment where the binding proceeds at two independent sites. The Scatchard plot for this binding model has a concave-upwards shape. When the concentration of the ribosomes is kept constant, a small systematic error in tRNA concentration changes this Scatchard plot markedly to a concave-downwards plot as though a co-operative interaction occurred. In contrast, when the tRNA concentration exceeds the ribosomal concentration and their concentration ratio is constant, the Scatchard plot is stable with respect to the systematic error. We suggest the latter type of experiment to be more appropriate. The results also imply a non-co-operative interaction of tRNAPhe with the 80 S ribosome.

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Gnanasundram, Sivakumar Vadivel, Isabelle C. Kos-Braun, and Martin Koš. "At least two molecules of the RNA helicase Has1 are simultaneously present in pre-ribosomes during ribosome biogenesis." Nucleic Acids Research 47, no. 20 (September 12, 2019): 10852–64. http://dx.doi.org/10.1093/nar/gkz767.

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Abstract The RNA helicase Has1 is involved in the biogenesis of both small and large ribosomal subunits. How it performs these separate roles is not fully understood. Here we provide evidence that at least two molecules of Has1 are temporarily present at the same time in 90S pre-ribosomes. We identified multiple Has1 binding sites in the 18S, 5.8S and 25S rRNAs. We show that while the Has1 catalytic activity is not required for binding to 5.8S/25S region in pre-rRNA, it is essential for binding to 18S sites. After the cleavage of pre-rRNA at the A2 site, Has1 remains associated not only with pre-60S but, unexpectedly, also with pre-40S ribosomes. The recruitment to 90S/pre-40S and pre-60S ribosomes is mutually independent. Our data provides insight into how Has1 performs its separate functions in the synthesis of both ribosomal subunits.

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Bisant, David, and Jacob Maizel. "Identification of ribosome binding sites inEscherichia coliusing neural network models." Nucleic Acids Research 23, no. 9 (1995): 1632–39. http://dx.doi.org/10.1093/nar/23.9.1632.

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34

Rozanska, Agata, Ricarda Richter-Dennerlein, Joanna Rorbach, Fei Gao, Richard J. Lewis, Zofia M. Chrzanowska-Lightowlers, and Robert N. Lightowlers. "The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome." Biochemical Journal 474, no. 13 (June 13, 2017): 2145–58. http://dx.doi.org/10.1042/bcj20170256.

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Accurate assembly and maturation of human mitochondrial ribosomes is essential for synthesis of the 13 polypeptides encoded by the mitochondrial genome. This process requires the correct integration of 80 proteins, 1 mt (mitochondrial)-tRNA and 2 mt-rRNA species, the latter being post-transcriptionally modified at many sites. Here, we report that human ribosome-binding factor A (RBFA) is a mitochondrial RNA-binding protein that exerts crucial roles in mitoribosome biogenesis. Unlike its bacterial orthologue, RBFA associates mainly with helices 44 and 45 of the 12S rRNA in the mitoribosomal small subunit to promote dimethylation of two highly conserved consecutive adenines. Characterization of RBFA-depleted cells indicates that this dimethylation is not a prerequisite for assembly of the small ribosomal subunit. However, the RBFA-facilitated modification is necessary for completing mt-rRNA maturation and regulating association of the small and large subunits to form a functional monosome implicating RBFA in the quality control of mitoribosome formation.

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Sterk, Maaike, Cédric Romilly, and E. Gerhart H. Wagner. "Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites." Nucleic Acids Research 46, no. 8 (February 6, 2018): 4188–99. http://dx.doi.org/10.1093/nar/gky073.

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Weiner, Iddo, Noam Shahar, Pini Marco, Iftach Yacoby, and Tamir Tuller. "Solving the Riddle of the Evolution of Shine-Dalgarno Based Translation in Chloroplasts." Molecular Biology and Evolution 36, no. 12 (September 10, 2019): 2854–60. http://dx.doi.org/10.1093/molbev/msz210.

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Abstract Chloroplasts originated from an ancient cyanobacterium and still harbor a bacterial-like genome. However, the centrality of Shine–Dalgarno ribosome binding, which predominantly regulates proteobacterial translation initiation, is significantly decreased in chloroplasts. As plastid ribosomal RNA anti-Shine–Dalgarno elements are similar to their bacterial counterparts, these sites alone cannot explain this decline. By computational simulation we show that upstream point mutations modulate the local structure of ribosomal RNA in chloroplasts, creating significantly tighter structures around the anti-Shine–Dalgarno locus, which in-turn reduce the probability of ribosome binding. To validate our model, we expressed two reporter genes (mCherry, hydrogenase) harboring a Shine–Dalgarno motif in the Chlamydomonas reinhardtii chloroplast. Coexpressing them with a 16S ribosomal RNA, modified according to our model, significantly enhances mCherry and hydrogenase expression compared with coexpression with an endogenous 16S gene.

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Politz, Joan C., Laura B. Lewandowski, and Thoru Pederson. "Signal recognition particle RNA localization within the nucleolus differs from the classical sites of ribosome synthesis." Journal of Cell Biology 159, no. 3 (November 11, 2002): 411–18. http://dx.doi.org/10.1083/jcb.200208037.

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The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I–specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.

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Pickering, Becky M., Sally A. Mitchell, Keith A. Spriggs, Mark Stoneley, and Anne E. Willis. "Bag-1 Internal Ribosome Entry Segment Activity Is Promoted by Structural Changes Mediated by Poly(rC) Binding Protein 1 and Recruitment of Polypyrimidine Tract Binding Protein 1." Molecular and Cellular Biology 24, no. 12 (June 15, 2004): 5595–605. http://dx.doi.org/10.1128/mcb.24.12.5595-5605.2004.

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ABSTRACT We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.

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Nürenberg-Goloub, Elina, Holger Heinemann, Milan Gerovac, and Robert Tampé. "Ribosome recycling is coordinated by processive events in two asymmetric ATP sites of ABCE1." Life Science Alliance 1, no. 3 (June 2018): e201800095. http://dx.doi.org/10.26508/lsa.201800095.

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Ribosome recycling orchestrated by ABCE1 is a fundamental process in protein translation and mRNA surveillance, connecting termination with initiation. Beyond the plenitude of well-studied translational GTPases, ABCE1 is the only essential factor energized by ATP, delivering the energy for ribosome splitting via two nucleotide-binding sites by a yet unknown mechanism. Here, we define how allosterically coupled ATP binding and hydrolysis events in ABCE1 empower ribosome recycling. ATP occlusion in the low-turnover control site II promotes formation of the pre-splitting complex and facilitates ATP engagement in the high-turnover site I, which in turn drives the structural reorganization required for ribosome splitting. ATP hydrolysis and ensuing release of ABCE1 from the small subunit terminate the post-splitting complex. Thus, ABCE1 runs through an allosterically coupled cycle of closure and opening at both sites, consistent with a processive clamp model. This study delineates the inner mechanics of ABCE1 and reveals why various ABCE1 mutants lead to defects in cell homeostasis, growth, and differentiation.

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Jomaa, Ahmad, Nikhil Jain, Joseph H. Davis, James R. Williamson, Robert A. Britton, and Joaquin Ortega. "Functional domains of the 50S subunit mature late in the assembly process." Nucleic Acids Research 42, no. 5 (December 13, 2013): 3419–35. http://dx.doi.org/10.1093/nar/gkt1295.

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Abstract Despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. Here, we analyze the late stages of assembly of the 50S subunit using Bacillus subtilis cells depleted of RbgA, a highly conserved GTPase. We found that RbgA-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins L16, L27, L28, L33a, L35 and L36. Using a novel pulse labeling/quantitative mass spectrometry technique, we show that this particle is physiologically relevant and is capable of maturing into a complete 50S particle. Cryo-electron microscopy and chemical probing revealed that the central protuberance, the GTPase associating region and tRNA-binding sites in this intermediate are unstructured. These findings demonstrate that key functional sites of the 50S subunit remain unstructured until late stages of maturation, preventing the incomplete subunit from prematurely engaging in translation. Finally, structural and biochemical analysis of a ribosome particle depleted of L16 indicate that L16 binding is necessary for the stimulation of RbgA GTPase activity and, in turn, release of this co-factor, and for conversion of the intermediate to a complete 50S subunit.

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Dorner, S., N. Polacek, U. Schulmeister, C. Panuschka, and A. Barta. "Molecular aspects of the ribosomal peptidyl transferase." Biochemical Society Transactions 30, no. 6 (November 1, 2002): 1131–37. http://dx.doi.org/10.1042/bst0301131.

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The proteins in a living cell are synthesized on a large bipartite ribonucleoprotein complex termed the ribosome. The peptidyl transferase, which polymerizes amino acids to yield peptides, is localized on the large subunit. Biochemical investigations over the past 35 years have led to the hypothesis that rRNA has a major role in all ribosomal functions. The recent high resolution X-ray structures of the ribosomal subunits clearly demonstrated that peptidyl transfer is an RNA-mediated process. As all ribosomal activities are dependent on bivalent metal ions, as is the case for most ribozymes, we investigated metal-ion-binding sites in rRNA by metal-ion-cleavage reactions. Some cleavage sites are near active sites and are evolutionarily highly conserved. The structure of the active site is flexible and undergoes changes during translocation and activation of the ribosome. Using modified P-site substrates, we showed that the 2′-OH group of the terminal adenosine is important for peptidyl transfer. These substrates were also used to investigate the metal ion dependency of the peptidyl transferase reaction.

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Fremerey, Julia, Pavel Morozov, Cindy Meyer, Aitor Garzia, Marianna Teplova, Thomas Tuschl, and Arndt Borkhardt. "Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties." Blood 128, no. 22 (December 2, 2016): 5056. http://dx.doi.org/10.1182/blood.v128.22.5056.5056.

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Abstract Introduction Nucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis. Methods In order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed. Results This study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36. Conclusion This study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. Disclosures No relevant conflicts of interest to declare.

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Arenz, Stefan, Manuel F. Juette, Michael Graf, Fabian Nguyen, Paul Huter, Yury S. Polikanov, Scott C. Blanchard, and Daniel N. Wilson. "Structures of the orthosomycin antibiotics avilamycin and evernimicin in complex with the bacterial 70S ribosome." Proceedings of the National Academy of Sciences 113, no. 27 (June 21, 2016): 7527–32. http://dx.doi.org/10.1073/pnas.1604790113.

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The ribosome is one of the major targets for therapeutic antibiotics; however, the rise in multidrug resistance is a growing threat to the utility of our current arsenal. The orthosomycin antibiotics evernimicin (EVN) and avilamycin (AVI) target the ribosome and do not display cross-resistance with any other classes of antibiotics, suggesting that they bind to a unique site on the ribosome and may therefore represent an avenue for development of new antimicrobial agents. Here we present cryo-EM structures of EVN and AVI in complex with the Escherichia coli ribosome at 3.6- to 3.9-Å resolution. The structures reveal that EVN and AVI bind to a single site on the large subunit that is distinct from other known antibiotic binding sites on the ribosome. Both antibiotics adopt an extended conformation spanning the minor grooves of helices 89 and 91 of the 23S rRNA and interacting with arginine residues of ribosomal protein L16. This binding site overlaps with the elbow region of A-site bound tRNA. Consistent with this finding, single-molecule FRET (smFRET) experiments show that both antibiotics interfere with late steps in the accommodation process, wherein aminoacyl-tRNA enters the peptidyltransferase center of the large ribosomal subunit. These data provide a structural and mechanistic rationale for how these antibiotics inhibit the elongation phase of protein synthesis.

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Salis, Howard M., Ethan A. Mirsky, and Christopher A. Voigt. "Automated design of synthetic ribosome binding sites to control protein expression." Nature Biotechnology 27, no. 10 (October 2009): 946–50. http://dx.doi.org/10.1038/nbt.1568.

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Choi, Junhong, and Joseph D. Puglisi. "Three tRNAs on the ribosome slow translation elongation." Proceedings of the National Academy of Sciences 114, no. 52 (December 11, 2017): 13691–96. http://dx.doi.org/10.1073/pnas.1719592115.

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During protein synthesis, the ribosome simultaneously binds up to three different transfer RNA (tRNA) molecules. Among the three tRNA binding sites, the regulatory role of the exit (E) site, where deacylated tRNA spontaneously dissociates from the translational complex, has remained elusive. Here we use two donor–quencher pairs to observe and correlate both the conformation of ribosomes and tRNAs as well as tRNA occupancy. Our results reveal a partially rotated state of the ribosome wherein all three tRNA sites are occupied during translation elongation. The appearance and lifetime of this state depend on the E-site tRNA dissociation kinetics, which may vary among tRNA species and depends on temperature and ionic strength. The 3-tRNA partially rotated state is not a proper substrate for elongation factor G (EF-G), thus inhibiting translocation until the E-site tRNA dissociates. Our result presents two parallel kinetic pathways during translation elongation, underscoring the ability of E-site codons to modulate the dynamics of protein synthesis.

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Wower, Jacek, Iwona K. Wower, Stanislav V. Kirillov, Kirill V. Rosen, Robert A. Zimmermann, and Stephen S. Hixson. "Peptidyl transferase and beyond." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1041–47. http://dx.doi.org/10.1139/o95-111.

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The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA. Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined. Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome. In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3′ end of tRNA at the A, P, and E sites. In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs. In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center.Key words: peptidyl transferase, ribosome, tRNA, photoreactive nucleos/tides, crosslinking.

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Romilly, Cédric, Sebastian Deindl, and E. Gerhart H. Wagner. "The ribosomal protein S1-dependent standby site in tisB mRNA consists of a single-stranded region and a 5′ structure element." Proceedings of the National Academy of Sciences 116, no. 32 (July 18, 2019): 15901–6. http://dx.doi.org/10.1073/pnas.1904309116.

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In bacteria, stable RNA structures that sequester ribosome-binding sites (RBS) impair translation initiation, and thus protein output. In some cases, ribosome standby can overcome inhibition by structure: 30S subunits bind sequence-nonspecifically to a single-stranded region and, on breathing of the inhibitory structure, relocate to the RBS for initiation. Standby can occur over long distances, as in the active, +42 tisB mRNA, encoding a toxin. This mRNA is translationally silenced by an antitoxin sRNA, IstR-1, that base pairs to the standby site. In tisB and other cases, a direct interaction between 30S subunits and a standby site has remained elusive. Based on fluorescence anisotropy experiments, ribosome toeprinting results, in vitro translation assays, and cross-linking–immunoprecipitation (CLIP) in vitro, carried out on standby-proficient and standby-deficient tisB mRNAs, we provide a thorough characterization of the tisB standby site. 30S subunits and ribosomal protein S1 alone display high-affinity binding to standby-competent fluorescein-labeled +42 mRNA, but not to mRNAs that lack functional standby sites. Ribosomal protein S1 is essential for standby, as 30∆S1 subunits do not support standby-dependent toeprints and TisB translation in vitro. S1 alone- and 30S-CLIP followed by RNA-seq mapping shows that the functional tisB standby site consists of the expected single-stranded region, but surprisingly, also a 5′-end stem-loop structure. Removal of the latter by 5′-truncations, or disruption of the stem, abolishes 30S binding and standby activity. Based on the CLIP-read mapping, the long-distance standby effect in +42 tisB mRNA (∼100 nt) is tentatively explained by S1-dependent directional unfolding toward the downstream RBS.

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Walters, Beth, Armend Axhemi, Eckhard Jankowsky, and Sunnie R. Thompson. "Binding of a viral IRES to the 40S subunit occurs in two successive steps mediated by eS25." Nucleic Acids Research 48, no. 14 (July 1, 2020): 8063–73. http://dx.doi.org/10.1093/nar/gkaa547.

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Abstract The mechanism for how internal ribosome entry sites (IRESs) recruit ribosomes to initiate translation of an mRNA is not completely understood. We investigated how a 40S subunit was recruited by the cricket paralysis virus intergenic region (CrPV IGR) IRES to form a stable 40S–IRES complex. Kinetic binding studies revealed that formation of the complex between the CrPV IGR and the 40S subunit consisted of two-steps: an initial fast binding step of the IRES to the 40S ribosomal subunit, followed by a slow unimolecular reaction consistent with a conformational change that stabilized the complex. We further showed that the ribosomal protein S25 (eS25), which is required by functionally and structurally diverse IRESs, impacts both steps of the complex formation. Mutations in eS25 that reduced CrPV IGR IRES activity either decreased 40S–IRES complex formation, or increased the rate of the conformational change that was required to form a stable 40S–IRES complex. Our data are consistent with a model in which eS25 facilitates initial binding of the CrPV IGR IRES to the 40S while ensuring that the conformational change stabilizing the 40S–IRES complex does not occur prematurely.

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Jogl, Gerwald, Reza Khayat, Eileen Murphy, Torsten Kleffmann, Kavindra Singh, Barbara Murray, and Kurt Krause. "164. Reporting the High-resolution Structure of the Enterococcal Ribosome: A New Template for Antibiotic Discovery." Open Forum Infectious Diseases 5, suppl_1 (November 2018): S15—S16. http://dx.doi.org/10.1093/ofid/ofy209.034.

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Abstract Background The ribosome is a rich target for antibiotic design and its structural secrets have been described at the atomic level over the past 2 decades. However, most bacterial ribosome structures come from nonpathogenic species of Archaea or thermophilic bacteria. To aid in the development of modern antibiotics against the enterococcus, we report the structure of the ribosome from Enterococcus faecalis at 3.5 Å resolution using cryo-electron microscopy. Methods E. faecalis strain OG1 was grown in liquid culture, collected and lysed using a French press. 70S ribosomes were purified using centrifugation through a sucrose cushion followed by column chromatography and sucrose gradient centrifugation. 70S particles were diluted in buffer and applied to a holey carbon grid and using an FEI vitrobot were flash-frozen in liquid ethane. Data were collected on an FEI Titan Krios operating at 300 kV acceleration voltage. The particles classified into 6 distinct structures based on their composition. Completed maps were utilized for structure modelling using Coot and were then refined using real space refinement within Phenix. Results High-quality maps of the 70S ribosome were obtained at up to 3.5 Å resolution in several distinct conformations. The 23S, 16S, and 5S RNA structures were almost completely built into maps with clear density. All but 2 ribosome proteins L25 and L33 have been placed in density. The A, P, and E sites were built into unambiguous density and found to be consistent with other bacterial structures. Notably, 1 EM density map contains an uncharged t-RNA molecule in the E site. The sites identified for current antibiotics are also well defined and interpretable. This 70S structural platform is suitable for structural analysis of antibiotic binding sites, especially for those antibiotics directed specifically against the enterococcal ribosome. Conclusion For the first time, the structure of the ribosome from the important human pathogen Enterococcus faecalis has been determined. The maps were obtained at high resolution and found to be suitable for antibiotic design. It is anticipated that the continued determination of the structures of ribosomes from pathogens will aid in the discovery of new treatments for infectious diseases. Disclosures All authors: No reported disclosures.

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Anand, Monika, Bharvi Balar, Rory Ulloque, Stephane R. Gross, and Terri Goss Kinzy. "Domain and Nucleotide Dependence of the Interaction between Saccharomyces cerevisiae Translation Elongation Factors 3 and 1A." Journal of Biological Chemistry 281, no. 43 (September 5, 2006): 32318–26. http://dx.doi.org/10.1074/jbc.m601899200.

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Eukaryotic translation elongation factor 3 (eEF3) is a fungal-specific ATPase proposed to catalyze the release of deacylated-tRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775–980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperature-sensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.

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Journal articles on the topic 'Ribosome binding sites'

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