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1
Yang, Rui, Luis R. Cruz-Vera, and Charles Yanofsky. "23S rRNA Nucleotides in the Peptidyl Transferase Center Are Essential for Tryptophanase Operon Induction." Journal of Bacteriology 191, no. 11 (March 27, 2009): 3445–50. http://dx.doi.org/10.1128/jb.00096-09.
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ABSTRACT Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNAPro leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNAPro by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNAPro to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNAPro from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression.
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Cruz-Vera, Luis R., Aaron New, Catherine Squires, and Charles Yanofsky. "Ribosomal Features Essential for tna Operon Induction: Tryptophan Binding at the Peptidyl Transferase Center." Journal of Bacteriology 189, no. 8 (February 9, 2007): 3140–46. http://dx.doi.org/10.1128/jb.01869-06.
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ABSTRACT Features of the amino acid sequence of the TnaC nascent peptide are recognized by the translating ribosome. Recognition leads to tryptophan binding within the translating ribosome, inhibiting the termination of tnaC translation and preventing Rho-dependent transcription termination in the tna operon leader region. It was previously shown that inserting an adenine residue at position 751 or introducing the U2609C change in 23S rRNA or introducing the K90W replacement in ribosomal protein L22 prevented tryptophan induction of tna operon expression. It was also observed that an adenine at position 752 of 23S rRNA was required for induction. In the current study, the explanation for the lack of induction by these altered ribosomes was investigated. Using isolated TnaC-ribosome complexes, it was shown that although tryptophan inhibits puromycin cleavage of TnaC-tRNAPro with wild-type ribosome complexes, it does not inhibit cleavage with the four mutant ribosome complexes examined. Similarly, tryptophan prevents sparsomycin inhibition of TnaC-tRNAPro cleavage with wild-type ribosome complexes but not with these mutant ribosome complexes. Additionally, a nucleotide located close to the peptidyl transferase center, A2572, which was protected from methylation by tryptophan with wild-type ribosome complexes, was not protected with mutant ribosome complexes. These findings identify specific ribosomal residues located in the ribosome exit tunnel that recognize features of the TnaC peptide. This recognition creates a free tryptophan-binding site in the peptidyl transferase center, where bound tryptophan inhibits peptidyl transferase activity.
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Agmon, Ilana, Anat Bashan, and Ada Yonath. "On Ribosome Conservation and Evolution." Israel Journal of Ecology and Evolution 52, no. 3-4 (April 12, 2006): 359–74. http://dx.doi.org/10.1560/ijee_52_3-4_359.
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The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A-> P-site passage of the tRNA terminus in the peptidyl transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC, the rotatory motion leads to stereochemistry suitable for peptide bond formation, as well as for substrate-mediated catalysis, consistent with quantum mechanical calculations elucidating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant PTC mobility and supported the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, utilizing free amino acids. Genetic control of the reaction evolved after poly-peptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotide orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures.
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Porse, Bo T., Cristina Rodriguez-Fonseca, Ilia Leviev, and Roger A. Garrett. "Antibiotic inhibition of the movement of tRNA substrates through a peptidyl transferase cavity." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 877–85. http://dx.doi.org/10.1139/o95-095.
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The present review attempts to deal with movement of tRNA substrates through the peptidyl transferase centre on the large ribosomal subunit and to explain how this movement is interrupted by antibiotics. It builds on the concept of hybrid tRNA states forming on ribosomes and on the observed movement of the 5′ end of P-site-bound tRNA relative to the ribosome that occurs on peptide bond formation. The 3′ ends of the tRNAs enter, and move through, a catalytic cavity where antibiotics are considered to act by at least three primary mechanisms: (i) they interfere with the entry of the aminoacyl moiety into the catalytic cavity before peptide bond formation; (ii) they inhibit movement of the nascent peptide along the peptide channel, a process that may generally involve destabilization of the peptidyl tRNA, and (iii) they prevent movement of the newly deacylated tRNA between the P/P and hybrid P/E sites on peptide bond formation.Key words: peptidyl transferase cavity, transient tRNA states, antibiotics, inhibitory mechanism, subunit–subunit interactions.
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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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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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Agmon, Ilana. "Hypothesis: Spontaneous Advent of the Prebiotic Translation System via the Accumulation of L-Shaped RNA Elements." International Journal of Molecular Sciences 19, no. 12 (December 12, 2018): 4021. http://dx.doi.org/10.3390/ijms19124021.
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The feasibility of self-assembly of a translation system from prebiotic random RNA chains is a question that is central to the ability to conceive life emerging by natural processes. The spontaneous materialization of a translation system would have required the autonomous formation of proto-transfer RNA (tRNA) and proto-ribosome molecules that are indispensable for translating an RNA chain into a polypeptide. Currently, the vestiges of a non-coded proto-ribosome, which could have only catalyzed the formation of a peptide bond between random amino acids, is consensually localized in the region encircling the peptidyl transferase center of the ribosomal large subunit. The work presented here suggests, based on high resolution structures of ribosomes complexed with messenger RNA (mRNA) and tRNAs, that three types of L-shaped RNA building blocks derived from the modern ribosome, alongside with an L-shaped proto-tRNA, each composed of about 70-mer, could have randomly occurred in the prebiotic world and combined to form a simple translation system. The model of the initial coded proto-ribosome, which includes the active sites of both ribosomal subunits, together with a bridging element, incorporates less than 6% of the current prokaryotic rRNA, yet it integrates all of the ribosomal components that are vital for synthesizing the earliest coded polypeptides.
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Powers, Kyle T., Flint Stevenson-Jones, Sathish K. N. Yadav, Beate Amthor, Joshua C. Bufton, Ufuk Borucu, Dakang Shen, et al. "Blasticidin S inhibits mammalian translation and enhances production of protein encoded by nonsense mRNA." Nucleic Acids Research 49, no. 13 (June 22, 2021): 7665–79. http://dx.doi.org/10.1093/nar/gkab532.
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Abstract Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3′CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1’s accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.
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Marks, James, Krishna Kannan, Emily J. Roncase, Dorota Klepacki, Amira Kefi, Cédric Orelle, Nora Vázquez-Laslop, and Alexander S. Mankin. "Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center." Proceedings of the National Academy of Sciences 113, no. 43 (October 10, 2016): 12150–55. http://dx.doi.org/10.1073/pnas.1613055113.
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The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.
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Swaney, Steven, Mark McCroskey, Dean Shinabarger, Zhigang Wang, Benjamin A. Turner, and Christian N. Parker. "Characterization of a High-Throughput Screening Assay for Inhibitors of Elongation Factor P and Ribosomal Peptidyl Transferase Activity." Journal of Biomolecular Screening 11, no. 7 (September 14, 2006): 736–42. http://dx.doi.org/10.1177/1087057106291634.
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Elongation Factor P (EF-P) is an essential component of bacterial protein synthesis, enhancing the rate of translation by facilitating the addition of amino acids to the growing peptide chain. Using purified Staphylococcus aureus EF-P and a reconstituted Escherichia coli ribosomal system, an assay monitoring the addition of radiolabeled N-formyl methionine to biotinylated puromycin was developed. Reaction products were captured with streptavidin-coated scintillation proximity assay (SPA) beads and quantified by scintillation counting. Data from the assay were used to create a kinetic model of the reaction scheme. In this model, EF-P binding to the ribosome essentially doubled the rate of the ribosomal peptidyl transferase reaction. As described here, EF-P bound to the ribosomes with an apparent Ka of 0.75 μM, and the substrates N-fMet-tRNA and biotinylated puromycin had apparent Kms of 19 μM and 0.5 μM, respectively. The assay was shown to be sensitive to a number of antibiotics known to target ribosomal peptide bond synthesis, such as chloramphenicol and puromycin, but not inhibitors that target other stages of protein synthesis, such as fusidic acid or thiostrepton.
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d’Aquino, Anne E., Tasfia Azim, Nikolay A. Aleksashin, Adam J. Hockenberry, Antje Krüger, and Michael C. Jewett. "Mutational characterization and mapping of the 70S ribosome active site." Nucleic Acids Research 48, no. 5 (February 3, 2020): 2777–89. http://dx.doi.org/10.1093/nar/gkaa001.
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Abstract The synthetic capability of the Escherichia coli ribosome has attracted efforts to repurpose it for novel functions, such as the synthesis of polymers containing non-natural building blocks. However, efforts to repurpose ribosomes are limited by the lack of complete peptidyl transferase center (PTC) active site mutational analyses to inform design. To address this limitation, we leverage an in vitro ribosome synthesis platform to build and test every possible single nucleotide mutation within the PTC-ring, A-loop and P-loop, 180 total point mutations. These mutant ribosomes were characterized by assessing bulk protein synthesis kinetics, readthrough, assembly, and structure mapping. Despite the highly-conserved nature of the PTC, we found that >85% of the PTC nucleotides possess mutational flexibility. Our work represents a comprehensive single-point mutant characterization and mapping of the 70S ribosome's active site. We anticipate that it will facilitate structure-function relationships within the ribosome and make possible new synthetic biology applications.
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Petrov, Anton S., Burak Gulen, Ashlyn M. Norris, Nicholas A. Kovacs, Chad R. Bernier, Kathryn A. Lanier, George E. Fox, et al. "History of the ribosome and the origin of translation." Proceedings of the National Academy of Sciences 112, no. 50 (November 30, 2015): 15396–401. http://dx.doi.org/10.1073/pnas.1509761112.
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We present a molecular-level model for the origin and evolution of the translation system, using a 3D comparative method. In this model, the ribosome evolved by accretion, recursively adding expansion segments, iteratively growing, subsuming, and freezing the rRNA. Functions of expansion segments in the ancestral ribosome are assigned by correspondence with their functions in the extant ribosome. The model explains the evolution of the large ribosomal subunit, the small ribosomal subunit, tRNA, and mRNA. Prokaryotic ribosomes evolved in six phases, sequentially acquiring capabilities for RNA folding, catalysis, subunit association, correlated evolution, decoding, energy-driven translocation, and surface proteinization. Two additional phases exclusive to eukaryotes led to tentacle-like rRNA expansions. In this model, ribosomal proteinization was a driving force for the broad adoption of proteins in other biological processes. The exit tunnel was clearly a central theme of all phases of ribosomal evolution and was continuously extended and rigidified. In the primitive noncoding ribosome, proto-mRNA and the small ribosomal subunit acted as cofactors, positioning the activated ends of tRNAs within the peptidyl transferase center. This association linked the evolution of the large and small ribosomal subunits, proto-mRNA, and tRNA.
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Schuwirth, Barbara S., Maria A. Borovinskaya, Cathy W. Hau, Wen Zhang, Antón Vila-Sanjurjo, James M. Holton, and Jamie H. Doudna Cate. "Structures of the Bacterial Ribosome at 3.5 Å Resolution." Science 310, no. 5749 (November 3, 2005): 827–34. http://dx.doi.org/10.1126/science.1117230.
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We describe two structures of the intact bacterial ribosome from Escherichia coli determined to a resolution of 3.5 angstroms by x-ray crystallography. These structures provide a detailed view of the interface between the small and large ribosomal subunits and the conformation of the peptidyl transferase center in the context of the intact ribosome. Differences between the two ribosomes reveal a high degree of flexibility between the head and the rest of the small subunit. Swiveling of the head of the small subunit observed in the present structures, coupled to the ratchet-like motion of the two subunits observed previously, suggests a mechanism for the final movements of messenger RNA (mRNA) and transfer RNAs (tRNAs) during translocation.
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Campbell, Tracey L., Denis M. Daigle, and Eric D. Brown. "Characterization of the Bacillus subtilis GTPase YloQ and its role in ribosome function." Biochemical Journal 389, no. 3 (July 26, 2005): 843–52. http://dx.doi.org/10.1042/bj20041873.
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We present an analysis of the cellular phenotype and biochemical activity of a conserved bacterial GTPase of unknown function (YloQ and YjeQ in Bacillus subtilis and Escherichia coli respectively) using a collection of antibiotics of diverse mechanisms and chemical classes. We created a yloQ deletion strain, which exhibited a slow growth phenotype and formed chains of filamentous cells. Additionally, we constructed a conditional mutant in yloQ, where growth was dependent on inducible expression from a complementing copy of the gene. In phenotypic studies, depletion of yloQ sensitized cells to antibiotics that bind at the peptide channel or peptidyl transferase centre, providing the first chemical genetic evidence linking this GTPase to ribosome function. Additional experiments using these small-molecule probes in vitro revealed that aminoglycoside antibiotics severely affected a previously characterized ribosome-associated GTPase activity of purified, recombinant YjeQ from E. coli. None of the antibiotics tested competed with YjeQ for binding to 30 or 70 S ribosomes. A closer examination of YloQ depletion revealed that the polyribosome profiles were altered and that decreased expression of YloQ led to the accumulation of ribosomal subunits at the expense of intact 70 S ribosomes. The present study provides the first evidence showing that YloQ/YjeQ may be involved in several areas of cellular metabolism, including cell division and ribosome function.
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Rodnina, Marina V., Malte Beringer, and Wolfgang Wintermeyer. "Mechanism of peptide bond formation on the ribosome." Quarterly Reviews of Biophysics 39, no. 3 (August 2006): 203–25. http://dx.doi.org/10.1017/s003358350600429x.
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1. The ribosome 2042. Peptide bond formation is catalyzed by RNA 2053. Characteristics of the uncatalyzed reaction 2074. Potential catalytic strategies of the ribosome 2075. Experimental systems 2086. Substrate binding in the PT center 2107. Induced fit in the active site 2118. pH dependence of peptide bond formation 2129. Reaction with full-length aa-tRNA 21410. Role of active-site residues 21511. pH-dependent structural changes of the active site 21612. Entropic catalysis 21713. Role of 2′-OH of A76 in P-site tRNA 21814. Catalysis by proton shuttling 21915. Plasticity of the active site 22016. Conclusions 22117. Acknowledgments 22218. References 222Peptide bond formation is the fundamental reaction of ribosomal protein synthesis. The ribosome's active site – the peptidyl transferase center – is composed of rRNA, and thus the ribosome is the largest known RNA catalyst. The ribosome accelerates peptide bond formation by 107-fold relative to the uncatalyzed reaction. Recent progress of structural, biochemical and computational approaches has provided a fairly detailed picture of the catalytic mechanisms employed by the ribosome. Energetically, catalysis is entirely entropic, indicating an important role of solvent reorganization, substrate positioning, and/or orientation of the reacting groups within the active site. The ribosome provides a pre-organized network of electrostatic interactions that stabilize the transition state and facilitate proton shuttling involving ribose hydroxyl groups of tRNA. The catalytic mechanism employed by the ribosome suggests how ancient RNA-world enzymes may have functioned.
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Wang, Wei, Wanqiu Li, Xueliang Ge, Kaige Yan, Chandra Sekhar Mandava, Suparna Sanyal, and Ning Gao. "Loss of a single methylation in 23S rRNA delays 50S assembly at multiple late stages and impairs translation initiation and elongation." Proceedings of the National Academy of Sciences 117, no. 27 (June 22, 2020): 15609–19. http://dx.doi.org/10.1073/pnas.1914323117.
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Ribosome biogenesis is a complex process, and dozens of factors are required to facilitate and regulate the subunit assembly in bacteria. The 2′-O-methylation of U2552 in 23S rRNA by methyltransferase RrmJ is a crucial step in late-stage assembly of the 50S subunit. Its absence results in severe growth defect and marked accumulation of pre50S assembly intermediates. In the present work, we employed cryoelectron microscopy to characterize a set of late-stage pre50S particles isolated from anEscherichia coliΔrrmJstrain. These assembly intermediates (solved at 3.2 to 3.8 Å resolution) define a collection of late-stage particles on a progressive assembly pathway. Apart from the absence of L16, L35, and L36, major structural differences between these intermediates and the mature 50S subunit are clustered near the peptidyl transferase center, such as H38, H68-71, and H89-93. In addition, the ribosomal A-loop of the mature 50S subunit from ΔrrmJstrain displays large local flexibility on nucleotides next to unmethylated U2552. Fast kinetics-based biochemical assays demonstrate that the ΔrrmJ50S subunit is only 50% active and two times slower than the WT 50S subunit in rapid subunit association. While the ΔrrmJ70S ribosomes show no defect in peptide bond formation, peptide release, and ribosome recycling, they translocate with 20% slower rate than the WT ribosomes in each round of elongation. These defects amplify during synthesis of the full-length proteins and cause overall defect in protein synthesis. In conclusion, our data reveal the molecular roles of U2552 methylation in both ribosome biogenesis and protein translation.
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Bashan, A., and A. Yonath. "Ribosome crystallography: catalysis and evolution of peptide-bond formation, nascent chain elongation and its co-translational folding." Biochemical Society Transactions 33, no. 3 (June 1, 2005): 488–92. http://dx.doi.org/10.1042/bst0330488.
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A ribosome is a ribozyme polymerizing amino acids, exploiting positional- and substrate-mediated chemical catalysis. We showed that peptide-bond formation is facilitated by the ribosomal architectural frame, provided by a sizable symmetry-related region in and around the peptidyl transferase centre, suggesting that the ribosomal active site was evolved by gene fusion. Mobility in tunnel components is exploited for elongation arrest as well as for trafficking nascent proteins into the folding space bordered by the bacterial chaperone, namely the trigger factor.
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Bøsling, Jacob, Susan M. Poulsen, Birte Vester, and Katherine S. Long. "Resistance to the Peptidyl Transferase Inhibitor Tiamulin Caused by Mutation of Ribosomal Protein L3." Antimicrobial Agents and Chemotherapy 47, no. 9 (September 2003): 2892–96. http://dx.doi.org/10.1128/aac.47.9.2892-2896.2003.
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ABSTRACT The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the peptidyl transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the peptidyl transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.
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Erales, Jenny, Virginie Marchand, Baptiste Panthu, Sandra Gillot, Stéphane Belin, Sandra E. Ghayad, Maxime Garcia, et al. "Evidence for rRNA 2′-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes." Proceedings of the National Academy of Sciences 114, no. 49 (November 20, 2017): 12934–39. http://dx.doi.org/10.1073/pnas.1707674114.
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Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2′-O-methylation (2′-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2′-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2′-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2′-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2′-O-Me, we identified a repertoire of 2′-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2′-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2′-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2′-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.
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Lawrence, Marlon G., Lasse Lindahl, and Janice M. Zengel. "Effects on Translation Pausing of Alterations in Protein and RNA Components of the Ribosome Exit Tunnel." Journal of Bacteriology 190, no. 17 (June 27, 2008): 5862–69. http://dx.doi.org/10.1128/jb.00632-08.
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ABSTRACT Amino acids are polymerized into peptides in the peptidyl transferase center of the ribosome. The nascent peptides then pass through the exit tunnel before they reach the extraribosomal environment. A number of nascent peptides interact with the exit tunnel and stall elongation at specific sites within their peptide chain. Several mutational changes in RNA and protein components of the ribosome have previously been shown to interfere with pausing. These changes are localized in the narrowest region of the tunnel, near a constriction formed by ribosomal proteins L4 and L22. To expand our knowledge about peptide-induced pausing, we performed a comparative study of pausing induced by two peptides, SecM and a short peptide, CrbCmlA, that requires chloramphenicol as a coinducer of pausing. We analyzed the effects of 15 mutational changes in L4 and L22, as well as the effects of methylating nucleotide A2058 of 23S rRNA, a nucleotide previously implicated in pausing and located close to the L4-L22 constriction. Our results show that methylation of A2058 and most mutational changes in L4 and L22 have differential effects on pausing in response to CrbCmlA and SecM. Only one change, a 6-amino-acid insertion after amino acid 72 in L4, affects pausing in both peptides. We conclude that the two peptides interact with different regions of the exit tunnel. Our results suggest that either the two peptides use different mechanisms of pausing or they interact differently but induce similar inhibitory conformational changes in functionally important regions of the ribosome.
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Gribling-Burrer, Anne-Sophie, Marco Chiabudini, Ying Zhang, Zonghao Qiu, Mario Scazzari, Tina Wölfle, Daniel Wohlwend, and Sabine Rospert. "A dual role of the ribosome-bound chaperones RAC/Ssb in maintaining the fidelity of translation termination." Nucleic Acids Research 47, no. 13 (May 22, 2019): 7018–34. http://dx.doi.org/10.1093/nar/gkz334.
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Abstract The yeast ribosome-associated complex RAC and the Hsp70 homolog Ssb are anchored to the ribosome and together act as chaperones for the folding and co-translational assembly of nascent polypeptides. In addition, the RAC/Ssb system plays a crucial role in maintaining the fidelity of translation termination; however, the latter function is poorly understood. Here we show that the RAC/Ssb system promotes the fidelity of translation termination via two distinct mechanisms. First, via direct contacts with the ribosome and the nascent chain, RAC/Ssb facilitates the translation of stalling-prone poly-AAG/A sequences encoding for polylysine segments. Impairment of this function leads to enhanced ribosome stalling and to premature nascent polypeptide release at AAG/A codons. Second, RAC/Ssb is required for the assembly of fully functional ribosomes. When RAC/Ssb is absent, ribosome biogenesis is hampered such that core ribosomal particles are structurally altered at the decoding and peptidyl transferase centers. As a result, ribosomes assembled in the absence of RAC/Ssb bind to the aminoglycoside paromomycin with high affinity (KD = 76.6 nM) and display impaired discrimination between stop codons and sense codons. The combined data shed light on the multiple mechanisms by which the RAC/Ssb system promotes unimpeded biogenesis of newly synthesized polypeptides.
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Kalpaxis, Dimitrios L., Panagiotis Karahalios, and M. Papapetropoulou. "Changes in Ribosomal Activity of Escherichia coli Cells during Prolonged Culture in Sea Salts Medium." Journal of Bacteriology 180, no. 12 (June 15, 1998): 3114–19. http://dx.doi.org/10.1128/jb.180.12.3114-3119.1998.
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ABSTRACT The activity of ribosomes from a clinical isolate ofEscherichia coli, exposed to starvation for 7 days in sea salts medium, was investigated by measuring the kinetic parameters of ribosomal peptidyltransferase, by using the puromycin reaction as a model reaction. No alterations in the extent of peptide bond formation were observed during starvation. In contrast, a 50% reduction in thek max/Ks ratio could be seen after 24 h of starvation; an additional 6 days of starvation resulted in a progressive but less abrupt decline in thek max/Ks value. {k max is the apparent catalytic rate constant of peptidyl transferase, and Ks is the dissociation constant of the encounter complex between acetyl (Ac)[3H]Phe-tRNA-poly(U)-ribosome and puromycin.} Although the distribution of ribosomal particles remained constant, a substantial decrease in the number of ribosomes per starved cell and a clear decline in the ability of ribosomes to bind AcPhe-tRNA were observed, particularly during the first day of starvation. Further analysis indicated that rRNA in general, but especially 23S rRNA, was rapidly degraded during the starvation period. In addition, the L12/L7 molar ratio decreased from 1.5 to 1 during the initial phase of starvation (up to 24 h) but remained constant during the subsequent starvation period. Ribosomes isolated from 24-h-starved cells, when artificially depleted of L7/L12 protein and reconstituted with L7/L12 protein from mid-logarithmic-phase cells, regenerated an L12/L7 molar ratio of 1.5 and restored the peptidyltransferase activity to a substantial level. An analogous effect of reconstitution on the efficiency of ribosomes in binding AcPhe-tRNA was evident not only during the initial phase but throughout the starvation period.
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Morse, Justin C., Dylan Girodat, Benjamin J. Burnett, Mikael Holm, Roger B. Altman, Karissa Y. Sanbonmatsu, Hans-Joachim Wieden, and Scott C. Blanchard. "Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection." Proceedings of the National Academy of Sciences 117, no. 7 (February 5, 2020): 3610–20. http://dx.doi.org/10.1073/pnas.1904469117.
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The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.
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Su, Weixin, Veerendra Kumar, Yichen Ding, Rya Ero, Aida Serra, Benjamin Sian Teck Lee, Andrew See Weng Wong, et al. "Ribosome protection by antibiotic resistance ATP-binding cassette protein." Proceedings of the National Academy of Sciences 115, no. 20 (April 30, 2018): 5157–62. http://dx.doi.org/10.1073/pnas.1803313115.
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The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.
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Xiong, Liqun, Patricia Kloss, Stephen Douthwaite, Niels Møller Andersen, Steven Swaney, Dean L. Shinabarger, and Alexander S. Mankin. "Oxazolidinone Resistance Mutations in 23S rRNA ofEscherichia coli Reveal the Central Region of Domain V as the Primary Site of Drug Action." Journal of Bacteriology 182, no. 19 (October 1, 2000): 5325–31. http://dx.doi.org/10.1128/jb.182.19.5325-5331.2000.
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ABSTRACT Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction, we selected Escherichia colioxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis in the wild-type rRNA operon produced an oxazolidinone resistance phenotype, establishing that the G2032A substitution was the determinant of resistance. Engineered U and C substitutions at G2032, as well as a G2447-to-U mutation, also conferred resistance to oxazolidinone. All the characterized resistance mutations were clustered in the vicinity of the central loop of domain V of 23S rRNA, suggesting that this rRNA region plays a major role in the interaction of the drug with the ribosome. Although the central loop of domain V is an essential integral component of the ribosomal peptidyl transferase, oxazolidinones do not inhibit peptide bond formation, and thus these drugs presumably interfere with another activity associated with the peptidyl transferase center.
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Gebetsberger, Jennifer, Marek Zywicki, Andrea Künzi, and Norbert Polacek. "tRNA-Derived Fragments Target the Ribosome and Function as Regulatory Non-Coding RNA inHaloferax volcanii." Archaea 2012 (2012): 1–11. http://dx.doi.org/10.1155/2012/260909.
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Nonprotein coding RNA (ncRNA) molecules have been recognized recently as major contributors to regulatory networks in controlling gene expression in a highly efficient manner. These RNAs either originate from their individual transcription units or are processing products from longer precursor RNAs. For example, tRNA-derived fragments (tRFs) have been identified in all domains of life and represent a growing, yet functionally poorly understood, class of ncRNA candidates. Here we present evidence that tRFs from the halophilic archaeonHaloferax volcaniidirectly bind to ribosomes. In the presented genomic screen of the ribosome-associated RNome, a 26-residue-long fragment originating from the 5′ part of valine tRNA was by far the most abundant tRF. The Val-tRF is processed in a stress-dependent manner and was found to primarily target the small ribosomal subunitin vitroandin vivo. As a consequence of ribosome binding, Val-tRF reduces protein synthesis by interfering with peptidyl transferase activity. Therefore this tRF functions as ribosome-bound small ncRNA capable of regulating gene expression inH. volcaniiunder environmental stress conditions probably by fine tuning the rate of protein production.
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Cruz-Vera, Luis R., and Charles Yanofsky. "Conserved Residues Asp16 and Pro24 of TnaC-tRNAPro Participate in Tryptophan Induction of tna Operon Expression." Journal of Bacteriology 190, no. 14 (April 18, 2008): 4791–97. http://dx.doi.org/10.1128/jb.00290-08.
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ABSTRACT In Escherichia coli, interactions between the nascent TnaC-tRNAPro peptidyl-tRNA and the translating ribosome create a tryptophan binding site in the ribosome where bound tryptophan inhibits TnaC-tRNAPro cleavage. This inhibition delays ribosome release, thereby inhibiting Rho factor binding and action, resulting in increased tna operon transcription. Replacing Trp12 of TnaC with any other amino acid residue was previously shown to prevent tryptophan binding and induction of tna operon expression. Genome-wide comparisons of TnaC amino acid sequences identify Asp16 and Pro24, as well as Trp12, as highly conserved TnaC residues. Replacing these residues with other residues was previously shown to influence tryptophan induction of tna operon expression. In this study, in vitro analyses were performed to examine the potential roles of Asp16 and Pro24 in tna operon induction. Replacing Asp16 or Pro24 of TnaC of E. coli with other amino acids established that these residues are essential for free tryptophan binding and inhibition of TnaC-tRNAPro cleavage at the peptidyl transferase center. Asp16 and Pro24 are in fact located in spatial positions corresponding to critical residues of AAP, another ribosome regulatory peptide. Sparsomycin-methylation protection studies further suggested that segments of 23S RNA were arranged differently in ribosomes bearing TnaCs with either the Asp16Ala or the Pro24Ala change. Thus, features of the amino acid sequence of TnaC of the nascent TnaC-tRNAPro peptidyl-tRNA, in addition to the presence of Trp12, are necessary for the nascent peptide to create a tryptophan binding/inhibition site in the translating ribosome.
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Скобликов, Н. Э., and N. E. Skoblikov. "Поиск реликтовых рибонуклеотидных и аминокислотных последовательностей, игравших ключевую роль в формировании рибосомы и современного разнообразия белков." Mathematical Biology and Bioinformatics 10, no. 1 (April 8, 2015): 116–30. http://dx.doi.org/10.17537/2015.10.116.
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The study presents the results of analysis of protein sequence database for prokaryotic microorganisms, which revealed a conservative peptide sequence element of 11 amino acid residues in 20 loci of 16 functionally and phylogenetically differing conservative proteins from representatives of various taxa. This amino acid motif IKAVRELGLER is presumably one of the Last Universal Peptide Ancestors (LUPAs). A fragment of ribosomal RNA (part of the A-site including stems H92, H90 and H93 of the peptidyl transferase center, PTC) translated from one of the potential reading frames is likely to be a source of genetic information for this sequence. We define this m/rRNA fragment with a function of a template for LUPA synthesis as the Last Universal RiboNucleic Ancestor (LURNA). We assume that LURNA and the peptides translated from its sequence were a source of the modern diversity of peptides.
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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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Subramanian, Selvi, and Linda Bonen. "Rapid evolution in sequence and length of the nuclear-located gene for mitochondrial L2 ribosomal protein in cereals." Genome 49, no. 3 (March 1, 2006): 275–81. http://dx.doi.org/10.1139/g05-098.
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The L2 ribosomal protein is typically one of the most conserved proteins in the ribosome and is universally present in bacterial, archaeal, and eukaryotic cytosolic and organellar ribosomes. It is usually 260–270 amino acids long and its binding to the large-subunit ribosomal RNA near the peptidyl transferase center is mediated by a β-barrel RNA-binding domain with 10 β strands. In the diverse land plants Marchantia polymorpha (liverwort) and Oryza sativa (rice), the mitochondrial-encoded L2 ribosomal protein is about 500 amino acids long owing to a centrally located expansion containing the β3–β4 strand region. We have determined that, in wheat, the functional rpl2 gene has been trans ferred to the nucleus and much of the plant-specific internal insert has been deleted. Its mRNA is only 1.2 kb, and two expressed copies in wheat encode proteins of 318 and 319 amino acids, so they are considerably shorter than the maize nuclear-located rpl2 gene of 448 codons. Comparative sequence analysis of cereal mitochondrial L2 ribosomal proteins indicates that the mid region has undergone unexpectedly rapid evolution during the last 60 million years.Key words: mitochondria, ribosomal protein, plants, evolutionary gene transfer.
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Llano-Sotelo, Beatriz, Jack Dunkle, Dorota Klepacki, Wen Zhang, Prabhavathi Fernandes, Jamie H. D. Cate, and Alexander S. Mankin. "Binding and Action of CEM-101, a New Fluoroketolide Antibiotic That Inhibits Protein Synthesis." Antimicrobial Agents and Chemotherapy 54, no. 12 (December 2010): 4961–70. http://dx.doi.org/10.1128/aac.00860-10.
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ABSTRACT We characterized the mechanism of action and the drug-binding site of a novel ketolide, CEM-101, which belongs to the latest class of macrolide antibiotics. CEM-101 shows high affinity for the ribosomes of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The ketolide shows high selectivity in its inhibitory action and readily interferes with synthesis of a reporter protein in the bacterial but not eukaryotic cell-free translation system. Binding of CEM-101 to its ribosomal target site was characterized biochemically and by X-ray crystallography. The X-ray structure of CEM-101 in complex with the E. coli ribosome shows that the drug binds in the major macrolide site in the upper part of the ribosomal exit tunnel. The lactone ring of the drug forms hydrophobic interactions with the walls of the tunnel, the desosamine sugar projects toward the peptidyl transferase center and interacts with the A2058/A2509 cleft, and the extended alkyl-aryl arm of the drug is oriented down the tunnel and makes contact with a base pair formed by A752 and U2609 of the 23S rRNA. The position of the CEM-101 alkyl-aryl extended arm differs from that reported for the side chain of the ketolide telithromycin complexed with either bacterial (Deinococcus radiodurans) or archaeal (Haloarcula marismortui) large ribosomal subunits but closely matches the position of the side chain of telithromycin complexed to the E. coli ribosome. A difference in the chemical structure of the side chain of CEM-101 in comparison with the side chain of telithromycin and the presence of the fluorine atom at position 2 of the lactone ring likely account for the superior activity of CEM-101. The results of chemical probing suggest that the orientation of the CEM-101 extended side chain observed in the E. coli ribosome closely resembles its placement in Staphylococcus aureus ribosomes and thus likely accurately reflects interaction of CEM-101 with the ribosomes of the pathogenic bacterial targets of the drug. Chemical probing further demonstrated weak binding of CEM-101, but not of erythromycin, to the ribosome dimethylated at A2058 by the action of Erm methyltransferase.
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Marintchev, Assen, and Gerhard Wagner. "Translation initiation: structures, mechanisms and evolution." Quarterly Reviews of Biophysics 37, no. 3-4 (November 2004): 197–284. http://dx.doi.org/10.1017/s0033583505004026.
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Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.
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Gagnon, Matthieu G., Jinzhong Lin, David Bulkley, and Thomas A. Steitz. "Crystal structure of elongation factor 4 bound to a clockwise ratcheted ribosome." Science 345, no. 6197 (August 7, 2014): 684–87. http://dx.doi.org/10.1126/science.1253525.
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Elongation factor 4 (EF4/LepA) is a highly conserved guanosine triphosphatase translation factor. It was shown to promote back-translocation of tRNAs on posttranslocational ribosome complexes and to compete with elongation factor G for interaction with pretranslocational ribosomes, inhibiting the elongation phase of protein synthesis. Here, we report a crystal structure of EF4–guanosine diphosphate bound to the Thermus thermophilus ribosome with a P-site tRNA at 2.9 angstroms resolution. The C-terminal domain of EF4 reaches into the peptidyl transferase center and interacts with the acceptor stem of the peptidyl-tRNA in the P site. The ribosome is in an unusual state of ratcheting with the 30S subunit rotated clockwise relative to the 50S subunit, resulting in a remodeled decoding center. The structure is consistent with EF4 functioning either as a back-translocase or a ribosome sequester.
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Yakusheva, Alena, Olga Shulenina, Evgeny Pichkur, Alena Paleskava, Alexander Myasnikov, and Andrey Konevega. "Abstract P-29: Cryoem Study of the Inhibition of Bacterial Ribosomes by Madumycin II." International Journal of Biomedicine 11, Suppl_1 (June 1, 2021): S24—S25. http://dx.doi.org/10.21103/ijbm.11.suppl_1.p29.
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Background: The efficiency of widely used antibiotics is limited by continuous improvement of resistance mechanisms. Thus, the research of poorly studied drugs that have not received practical use until now becomes relevant again. Protein translation is one of the major targets for antibiotics. Madumycin II (MADU) is an antibiotic of the streptogramin A class that binds to the peptidyl transferase center of the initiated bacterial 70S ribosome inhibiting the first cycle of peptide bond formation (I.A. Osterman et al. Nucleic Acids Res., 2017). The ability of MADU to interfere with translating ribosome is an open question that we address by investigation of high-resolution cryo-EM structures of MADU bound 70S ribosome complexes from Escherichia coli. Methods: Purified initiated and translating ribosome complexes preincubated with MADU were applied onto freshly glow discharged carbon-coated grids (Quantifoil R 1.2/1.3) and flash-frozen in the liquid ethane pre-cooled by liquid nitrogen in the Vitrobot Mark IV. Frozen grids were transferred into an in-house Titan Krios microscope. Data were collected using EPU software. Movie stacks were preprocessed in Warp software. For image processing, we have used several software packages: Relion 3.1, CryoSPARC, and CisTEM. The model was built in Coot. Results: We have obtained high-resolution cryo-EM structures of two ribosomal complexes with MADU before and after the first cycle of peptide bond formation with an average resolution of 2.3 Å. Preliminary analysis of the structures shows no major differences in the MADU binding mode to the ribosomal complexes under study suggesting that the quantity of amino acid residues attached to the P-site tRNA does not impact MADU bonding. Moreover, in both cases, we observed similar destabilization of the CCA-ends of A- and P-site tRNAs underlining the comparable influence of MADU on the ribosomal complexes. Conclusion: Our results suggest that although MADU binding site is located in the peptidyl transferase center, the presence of the second amino acid residue on the P-site tRNA does not preclude antibiotic binding. We assume that further elongation of the polypeptide chain would not have any impact either. High conformational lability of the CCA-ends of tRNA at the A and P sites upon binding of MADU obviously plays an important role in the inhibition mechanism of the bacterial ribosome. The further structural and biochemical analysis will be necessary to shed more light on the detailed mechanism of MADU action.
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Houben, Edith N. G., Raz Zarivach, Bauke Oudega, and Joen Luirink. "Early encounters of a nascent membrane protein." Journal of Cell Biology 170, no. 1 (June 27, 2005): 27–35. http://dx.doi.org/10.1083/jcb.200503035.
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An unbiased photo–cross-linking approach was used to probe the “molecular path” of a growing nascent Escherichia coli inner membrane protein (IMP) from the peptidyl transferase center to the surface of the ribosome. The nascent chain was initially in proximity to the ribosomal proteins L4 and L22 and subsequently contacted L23, which is indicative of progression through the ribosome via the main ribosomal tunnel. The signal recognition particle (SRP) started to interact with the nascent IMP and to target the ribosome–nascent chain complex to the Sec–YidC complex in the inner membrane when maximally half of the transmembrane domain (TM) was exposed from the ribosomal exit. The combined data suggest a flexible tunnel that may accommodate partially folded nascent proteins and parts of the SRP and SecY. Intraribosomal contacts of the nascent chain were not influenced by the presence of a functional TM in the ribosome.
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Agmon, Ilana, Anat Bashan, Raz Zarivach, and Ada Yonath. "Symmetry at the active site of the ribosome: structural and functional implications." Biological Chemistry 386, no. 9 (September 1, 2005): 833–44. http://dx.doi.org/10.1515/bc.2005.098.
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Abstract The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3′-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3′-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2′-hydroxyl.
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Beringer, Malte, and Marina V. Rodnina. "The Ribosomal Peptidyl Transferase." Molecular Cell 26, no. 3 (May 2007): 311–21. http://dx.doi.org/10.1016/j.molcel.2007.03.015.
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Wang, S., H. Sakai, and M. Wiedmann. "NAC covers ribosome-associated nascent chains thereby forming a protective environment for regions of nascent chains just emerging from the peptidyl transferase center." Journal of Cell Biology 130, no. 3 (August 1, 1995): 519–28. http://dx.doi.org/10.1083/jcb.130.3.519.
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We demonstrate that nascent polypeptide-associated complex (NAC) is one of the first cytosolic factors that newly synthesized nascent chains encounter. When NAC is present, nascent chains are segregated from the cytosol until approximately 30 amino acids in length, a finding consistent with the well-documented protease resistance of short ribosome-associated nascent chains. When NAC is removed, the normally protected nascent chains are susceptible to proteolysis. Therefore NAC, by covering COOH-terminal segments of nascent chains on the ribosome, perhaps together with ribosomal proteins, forms a protective environment for regions of nascent chains just emerging from the peptidyl transferase center. Since NAC is not a core ribosomal protein, the emergence of nascent chains from the ribosome may be more dynamic than previously thought.
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Lacoux, Caroline, Ludivine Wacheul, Kritika Saraf, Nicolas Pythoud, Emmeline Huvelle, Sabine Figaro, Marc Graille, Christine Carapito, Denis L. J. Lafontaine, and Valérie Heurgué-Hamard. "The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis." Nucleic Acids Research 48, no. 21 (November 9, 2020): 12310–25. http://dx.doi.org/10.1093/nar/gkaa972.
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Abstract The Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination.
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Englander, Michael T., Joshua L. Avins, Rachel C. Fleisher, Bo Liu, Philip R. Effraim, Jiangning Wang, Klaus Schulten, Thomas S. Leyh, Ruben L. Gonzalez, and Virginia W. Cornish. "The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center." Proceedings of the National Academy of Sciences 112, no. 19 (April 27, 2015): 6038–43. http://dx.doi.org/10.1073/pnas.1424712112.
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The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.
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Sweeney, Rosemary, and Meng-Chao Yao. "An Intragenic Suppressor of Cold Sensitivity Identifies Potentially Interacting Bases in the Peptidyl Transferase Center of Tetrahymena rRNA." Genetics 149, no. 2 (June 1, 1998): 937–46. http://dx.doi.org/10.1093/genetics/149.2.937.
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Abstract Peptidyl transfer of a growing peptide on a ribosome-bound transfer RNA (tRNA) to an incoming amino acyl tRNA is the central step in translation, and it may be catalyzed primarily by the large subunit (LSU) ribosomal RNA (rRNA). Genetic and biochemical evidence suggests that the central loop of domain V of the LSU rRNA plays a direct role in peptidyl transfer. It was previously found that a single base change at a universally conserved site in this region of the Tetrahymena thermophila LSU rRNA confers anisomycin resistance (an-r) as well as extremely slow growth, cold sensitivity, and aberrant cell morphology. Because anisomycin specifically inhibits peptidyl transfer, possibly by interfering with tRNA binding, it is likely that this mutant rRNA is defective in efficiently completing one of these steps. In the present study, we have isolated an intragenic suppressor mutation located only three bases away from the original mutation that partially reverses the slow growth and cold-sensitive phenotypes. These data imply that the functional interaction of these two bases is necessary for normal rRNA function, perhaps for peptidyl transfer or tRNA binding. These data provide the first demonstration of a functional interaction between bases within this rRNA region.
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Chancey, Scott T., Xiaoliu Zhou, Dorothea Zähner, and David S. Stephens. "Induction of Efflux-Mediated Macrolide Resistance in Streptococcus pneumoniae." Antimicrobial Agents and Chemotherapy 55, no. 7 (May 2, 2011): 3413–22. http://dx.doi.org/10.1128/aac.00060-11.
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ABSTRACTThe antimicrobial efflux system encoded by the operonmef(E)-melon the mobile genetic element MEGA inStreptococcus pneumoniaeand other Gram-positive bacteria is inducible by macrolide antibiotics and antimicrobial peptides. Induction may affect the clinical response to the use of macrolides. We developedmef(E)reporter constructs and a disk diffusion induction and resistance assay to determine the kinetics and basis ofmef(E)-melinduction. Induction occurred rapidly, with a >15-fold increase in transcription within 1 h of exposure to subinhibitory concentrations of erythromycin. A spectrum of environmental conditions, including competence and nonmacrolide antibiotics with distinct cellular targets, did not inducemef(E).Using 16 different structurally defined macrolides, induction was correlated with the amino sugar attached to C-5 of the macrolide lactone ring, not with the size (e.g., 14-, 15- or 16-member) of the ring or with the presence of the neutral sugar cladinose at C-3. Macrolides with a monosaccharide attached to C-5, known to block exit of the nascent peptide from the ribosome after the incorporation of up to eight amino acids, inducedmef(E)expression. Macrolides with a C-5 disaccharide, which extends the macrolide into the ribosomal exit tunnel, disrupting peptidyl transferase activity, did not induce it. The induction ofmef(E)did not require macrolide efflux, but the affinity of macrolides for the ribosome determined the availability for efflux and pneumococcal susceptibility. The induction ofmef(E)-melexpression by inducing macrolides appears to be based on specific interactions of the macrolide C-5 saccharide with the ribosome that alleviate transcriptional attenuation ofmef(E)-mel.
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Beringer, Malte, Christian Bruell, Liqun Xiong, Peter Pfister, Peter Bieling, Vladimir I. Katunin, Alexander S. Mankin, Erik C. Böttger, and Marina V. Rodnina. "Essential Mechanisms in the Catalysis of Peptide Bond Formation on the Ribosome." Journal of Biological Chemistry 280, no. 43 (August 29, 2005): 36065–72. http://dx.doi.org/10.1074/jbc.m507961200.
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Peptide bond formation is the main catalytic function of the ribo-some. The mechanism of catalysis is presumed to be highly conserved in all organisms. We tested the conservation by comparing mechanistic features of the peptidyl transfer reaction on ribosomes from Escherichia coli and the Gram-positive bacterium Mycobacterium smegmatis. In both cases, the major contribution to catalysis was the lowering of the activation entropy. The rate of peptide bond formation was pH independent with the natural substrate, amino-acyl-tRNA, but was slowed down 200-fold with decreasing pH when puromycin was used as a substrate analog. Mutation of the conserved base A2451 of 23 S rRNA to U did not abolish the pH dependence of the reaction with puromycin in M. smegmatis, suggesting that A2451 did not confer the pH dependence. However, the A2451U mutation alters the structure of the peptidyl transferase center and changes the pattern of pH-dependent rearrangements, as probed by chemical modification of 23 S rRNA. A2451 seems to function as a pivot point in ordering the structure of the peptidyl transferase center rather than taking part in chemical catalysis.
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Cooperman, Barry S., Tammy Wooten, Robert R. Traut, and Daniel P. Romero. "Histidine 229 in protein L2 is apparently essential for 50S peptidyl transferase activity." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1087–94. http://dx.doi.org/10.1139/o95-117.
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It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the work reported here, we reconstitute 50S subunits in which the H229Q variant of L2 replaces L2, with all other ribosomal components remaining unchanged, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserved His 229 to Gin results in a complete loss of peptidyl transferase activity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activity. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identical with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S subunits. Furthermore, as shown by chemical footprinting analysis, H229Q-L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, the effect of H229 mutation appears to be confined to an effect on peptidyl transferase activity, providing the most direct evidence for protein involvement in this function to date.Key words: protein L2, site-specific mutagenesis, peptidyl transferase, reconstitution, histidine.
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Wei, J., C. Wu, and M. S. Sachs. "The Arginine Attenuator Peptide Interferes with the Ribosome Peptidyl Transferase Center." Molecular and Cellular Biology 32, no. 13 (April 16, 2012): 2396–406. http://dx.doi.org/10.1128/mcb.00136-12.
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Cheng, Lin, Nian Hong, Xiang Qun Xu, Jie Yang, and You Quan Zhong. "A Competitive Model Reaction for the Ribosomal Peptide Bond Formation Catalyzed by Peptidyl Transferase." Applied Mechanics and Materials 496-500 (January 2014): 17–20. http://dx.doi.org/10.4028/www.scientific.net/amm.496-500.17.
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In this work, a series of theoretical methods were employed to investigate the reaction mechanisms of ribosomal peptide bond formation catalyzed by peptidyl transferase. For the studies described in this paper, reaction pathways and free energy barriers for the model reaction of the peptide bond synthesis were studied by performing Ab initio calculation. Two self-consistent reaction field (SCRF) methods were used to calculate of the whole reaction pathway. These results show that the present theoretical reaction mechanism is a potential and competitive one for the reaction modeling of the ribosomal peptide synthesis.
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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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Yan, Kang, Lenore Madden, Anthony E. Choudhry, Christine S. Voigt, Robert A. Copeland, and Richard R. Gontarek. "Biochemical Characterization of the Interactions of the Novel Pleuromutilin Derivative Retapamulin with Bacterial Ribosomes." Antimicrobial Agents and Chemotherapy 50, no. 11 (August 28, 2006): 3875–81. http://dx.doi.org/10.1128/aac.00184-06.
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ABSTRACT Retapamulin is a semisynthetic pleuromutilin derivative being developed as a topical antibiotic for treating bacterial infections of the skin. It is potent in vitro against susceptible and multidrug-resistant organisms commonly associated with bacterial skin infections. We report detailed mode of action studies demonstrating that retapamulin binds to the bacterial ribosome with high affinity, inhibits ribosomal peptidyl transferase activity, and partially inhibits the binding of the initiator tRNA substrate to the ribosomal P-site. Taken together, these data distinguish the mode of action of retapamulin from that of other classes of antibiotics. This unique mode of action may explain the lack of clinically relevant, target-specific cross-resistance of retapamulin with antibacterials in current use.
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Baßler, Jochen, Helge Paternoga, Iris Holdermann, Matthias Thoms, Sander Granneman, Clara Barrio-Garcia, Afua Nyarko, et al. "A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation." Journal of Cell Biology 207, no. 4 (November 17, 2014): 481–98. http://dx.doi.org/10.1083/jcb.201408111.
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Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.
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Chen, Chih-Wei, Julia A. Pavlova, Dmitrii A. Lukianov, Andrey G. Tereshchenkov, Gennady I. Makarov, Zimfira Z. Khairullina, Vadim N. Tashlitsky, et al. "Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome." Antibiotics 10, no. 4 (April 5, 2021): 390. http://dx.doi.org/10.3390/antibiotics10040390.
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Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.