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  • 1.
    Chan, Sherwin
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Frasch, Alejandra
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ch'ng, Jun-Hong
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden.;Natl Univ Singapore, Dept Microbiol, Singapore 117545, Singapore..
    del Pilar Quintana, Maria
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden.;Univ Rosario, Fac Ciencias Nat & Matemat, Escuela Med & Ciencias Salud, Calle 12C 6-25, Bogota, Colombia..
    Vesterlund, Mattias
    Karolinska Inst, Dept Oncol Pathol, Canc Prote, S-17176 Stockholm, Sweden..
    Ghorbal, Mehdi
    Univ Montpellier, Lab Parasitol Mycol, Fac Med, F-34090 Montpellier, France.;Univ Montpellier, UMR MiVEGEC, IRD 224, CNRS 5290, Montpellier, France..
    Joannin, Nicolas
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Franzen, Oscar
    Icahn Sch Med Mt Sinai, Inst Genom & Multiscale Biol, Dept Genet & Genom Sci, New York, NY 10029 USA..
    Lopez-Rubio, Jose-Juan
    Univ Montpellier, Lab Parasitol Mycol, Fac Med, F-34090 Montpellier, France.;Univ Montpellier, UMR MiVEGEC, IRD 224, CNRS 5290, Montpellier, France..
    Barbieri, Sonia
    Univ Svizzera Italiana, Inst Res Biomed, CH-6500 Bellinzona, Switzerland..
    Lanzavecchia, Antonio
    Univ Svizzera Italiana, Inst Res Biomed, CH-6500 Bellinzona, Switzerland.;ETH, Inst Microbiol, CH-8093 Zurich, Switzerland..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wahlgren, Mats
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol MTC, Box 280,Nobels Vag 16, S-17177 Stockholm, Sweden..
    Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor2017In: Nature Microbiology, E-ISSN 2058-5276, Vol. 2, no 7, article id 17068Article in journal (Refereed)
    Abstract [en]

    Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.

  • 2.
    Degiacomi, Giulia
    et al.
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Personne, Yoann
    Queen Mary Univ London, London E1 2AD, England.;UCL, Div Infect & Immun, Ctr Clin Microbiol, London, England..
    Mondesert, Guillaume
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hartkoorn, Ruben C.
    Ecole Polytech Fed Lausanne, Global Hlth Inst, Lausanne, Switzerland.;Univ Lille Nord France, Inst Pasteur Lille, Ctr Infect & Immun Lille, INSERM,CNRS,UMR 8204,U1019, Lille, France..
    Boldrin, Francesca
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Goel, Pavitra
    Queen Mary Univ London, London E1 2AD, England..
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Benjak, Andrej
    Ecole Polytech Fed Lausanne, Global Hlth Inst, Lausanne, Switzerland..
    Barrio, Maria Belen
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Ventura, Marcello
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Brown, Amanda C.
    Queen Mary Univ London, London E1 2AD, England.;Cornell Univ, Dept Microbiol & Immunol, Ithaca, NY 14853 USA..
    Leblanc, Veronique
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Bauer, Armin
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Cole, Stewart T.
    Ecole Polytech Fed Lausanne, Global Hlth Inst, Lausanne, Switzerland..
    Lagrange, Sophie
    Sanofi Aventis R&D, Drug Disposit, F-69367 Lyon, France..
    Parish, Tanya
    Queen Mary Univ London, London E1 2AD, England.;Infect Dis Res Inst, TB Discovery Res, Seattle, WA 98102 USA..
    Manganelli, Riccardo
    Univ Padua, Dept Mol Med, Via Gabelli 63, I-35121 Padua, Italy..
    Micrococcin P1-A bactericidal thiopeptide active against Mycobacterium tuberculosis2016In: Tuberculosis, ISSN 1472-9792, E-ISSN 1873-281X, Vol. 100, p. 95-101Article in journal (Refereed)
    Abstract [en]

    The lack of proper treatment for serious infectious diseases due to the emergence of multidrug resistance reinforces the need for the discovery of novel antibiotics. This is particularly true for tuberculosis (TB) for which 3.7% of new cases and 20% of previously treated cases are estimated to be caused by multi-drug resistant strains. In addition, in the case of TB, which claimed 1.5 million lives in 2014, the treatment of the least complicated, drug sensitive cases is lengthy and disagreeable. Therefore, new drugs with novel targets are urgently needed to control resistant Mycobacterium tuberculosis strains. In this manuscript we report the characterization of the thiopeptide micrococcin P1 as an anti-tubercular agent. Our biochemical experiments show that this antibiotic inhibits the elongation step of protein synthesis in mycobacteria. We have further identified micrococcin resistant mutations in the ribosomal protein L11 (RplK); the mutations were located in the proline loop at the N-terminus. Reintroduction of the mutations into a clean genetic background, confirmed that they conferred resistance, while introduction of the wild type RplK allele into resistant strains re-established sensitivity. We also identified a mutation in the 23S rRNA gene. These data, in good agreement with previous structural studies suggest that also in M. tuberculosis micrococcin P1 functions by binding to the cleft between the 23S rRNA and the L11 protein loop, thus interfering with the binding of elongation factors Tu and G (EF-Tu and EF-G) and inhibiting protein translocation.

  • 3. Feng, Boya
    et al.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Guo, Qiang
    Wang, Jie
    Cao, Wei
    Li, Ningning
    Zhang, Yixiao
    Zhang, Yanqing
    Wang, Zhixin
    Wu, Jiawei
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lei, Jianlin
    Gao, Ning
    Structural and Functional Insights into the Mode of Action of a Universally Conserved Obg GTPase2014In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, no 5, p. e1001866-Article in journal (Refereed)
    Abstract [en]

    Obg proteins are a family of P-loop GTPases, conserved from bacteria to human. The Obg protein in Escherichia coli (ObgE) has been implicated in many diverse cellular functions, with proposed molecular roles in two global processes, ribosome assembly and stringent response. Here, using pre-steady state fast kinetics we demonstrate that ObgE is an anti-association factor, which prevents ribosomal subunit association and downstream steps in translation by binding to the 50S subunit. ObgE is a ribosome dependent GTPase; however, upon binding to guanosine tetraphosphate (ppGpp), the global regulator of stringent response, ObgE exhibits an enhanced interaction with the 50S subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits. Furthermore, our cryo-electron microscopy (cryo-EM) structure of the 50S? ObgE? GMPPNP complex indicates that the evolutionarily conserved N-terminal domain (NTD) of ObgE is a tRNA structural mimic, with specific interactions with peptidyl-transferase center, displaying a marked resemblance to Class I release factors. These structural data might define ObgE as a specialized translation factor related to stress responses, and provide a framework towards future elucidation of functional interplay between ObgE and ribosome-associated (p) ppGpp regulators. Together with published data, our results suggest that ObgE might act as a checkpoint in final stages of the 50S subunit assembly under normal growth conditions. And more importantly, ObgE, as a (p) ppGpp effector, might also have a regulatory role in the production of the 50S subunit and its participation in translation under certain stressed conditions. Thus, our findings might have uncovered an under-recognized mechanism of translation control by environmental cues.

  • 4.
    Fislage, Marcus
    et al.
    VIB VUB Ctr Struct Biol, Brussels, Belgium;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA;Vrije Univ Brussel, Struct Biol Brussels, Brussels, Belgium.
    Zhang, Jingji
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Brown, Zuben Patrick
    Osaka Univ, Inst Prot Res, Lab Prot Synth & Express, Osaka, Japan;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Frank, Joachim
    Columbia Univ, Dept Biol Sci, New York, NY 10027 USA;Columbia Univ, Dept Biochem & Mol Biophys, New York, NY 10027 USA.
    Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-Tu(H84A)2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 11, p. 5861-5874Article in journal (Refereed)
    Abstract [en]

    The GTPase EF-Tu in ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) promotes rapid and accurate delivery of cognate aa-tRNAs to the ribosomal A site. Here we used cryo-EM to study the molecular origins of the accuracy of ribosome-aided recognition of a cognate ternary complex and the accuracy-amplifying role of themonitoring bases A1492, A1493 and G530 of the 16S rRNA. We used the GTPase-deficient EF-Tu variant H84A with native GTP, rather than non-cleavable GTP analogues, to trap a near-cognate ternary complex in high-resolution ribosomal complexes of varying codon-recognition accuracy. We found that ribosome complexes trapped by GTPase-deficicent ternary complex due to the presence of EF-TuH84A or non-cleavable GTP analogues have very similar structures. We further discuss speed and accuracy of initial aa-tRNA selection in terms of conformational changes of aa-tRNA and stepwise activation of the monitoring bases at the decoding center of the ribosome.

  • 5.
    Ge, Xueliang
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed)
    Abstract [en]

    The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

  • 6.
    Guo, Xiaohu
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bäckbro, Kristina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Koripella, Ravi Kiran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structure and function of FusB: an elongation factor G-binding fusidic acid resistance protein active in ribosomal translocation and recycling2012In: Open Biology, ISSN 2046-2441, Vol. 2, p. 120016-Article in journal (Refereed)
    Abstract [en]

    Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) to the ribosome after GTP hydrolysis during elongation and ribosome recycling. The plasmid pUB101-encoded protein FusB causes FA resistance in clinical isolates of Staphylococcus aureus through an interaction with EF-G. Here, we report 1.6 and 2.3 angstrom crystal structures of FusB. We show that FusB is a two-domain protein lacking homology to known structures, where the N-terminal domain is a four-helix bundle and the C-terminal domain has an alpha/beta fold containing a C4 treble clef zinc finger motif and two loop regions with conserved basic residues. Using hybrid constructs between S. aureus EF-G that binds to FusB and Escherichia coli EF-G that does not, we show that the sequence determinants for FusB recognition reside in domain IV and involve the C-terminal helix of S. aureus EF-G. Further, using kinetic assays in a reconstituted translation system, we demonstrate that FusB can rescue FA inhibition of tRNA translocation as well as ribosome recycling. We propose that FusB rescues S. aureus from FA inhibition by preventing formation or facilitating dissociation of the FA-locked EF-G-ribosome complex.

  • 7.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation2019In: eLIFE, E-ISSN 2050-084X, Vol. 8, article id e46124Article in journal (Refereed)
    Abstract [en]

    Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.

  • 8.
    Koripella, Ravi Kiran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Holm, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dourado, Daniel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Flores, Samuel
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A conserved histidine in switch-II of EF-G moderates release of inorganic phosphate2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12970Article in journal (Refereed)
    Abstract [en]

    Elongation factor G (EF-G), a translational GTPase responsible for tRNA-mRNA translocation possesses a conserved histidine (H91 in Escherichia coli) at the apex of switch-II, which has been implicated in GTPase activation and GTP hydrolysis. While H91A, H91R and H91E mutants showed different degrees of defect in ribosome associated GTP hydrolysis, H91Q behaved like the WT. However, all these mutants, including H91Q, are much more defective in inorganic phosphate (Pi) release, thereby suggesting that H91 facilitates Pi release. In crystal structures of the ribosome bound EF-G center dot GTP a tight coupling between H91 and the gamma-phosphate of GTP can be seen. Following GTP hydrolysis, H91 flips similar to 140 degrees in the opposite direction, probably with Pi still coupled to it. This, we suggest, promotes Pi to detach from GDP and reach the inter-domain space of EF-G, which constitutes an exit path for the Pi. Molecular dynamics simulations are consistent with this hypothesis and demonstrate a vital role of an Mg2+ ion in the process.

  • 9.
    Mandava, Chandra Sekhar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ederth, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kumar, Ranjeet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Szaflarski, Witold
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G2012In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 5, p. 2054-2064Article in journal (Refereed)
    Abstract [en]

    The ribosomal stalk in bacteria is composed of four or six copies of L12 proteins arranged in dimers that bind to the adjacent sites on protein L10, spanning 10 amino acids each from the L10 C-terminus. To study why multiple L12 dimers are required on the ribosome, we created a chromosomally engineered Escherichia coli strain, JE105, in which the peripheral L12 dimer binding site was deleted. Thus JE105 harbors ribosomes with only a single L12 dimer. Compared to MG1655, the parental strain with two L12 dimers, JE105 showed significant growth defect suggesting suboptimal function of the ribosomes with one L12 dimer. When tested in a cell-free reconstituted transcription-translation assay the synthesis of a full-length protein, firefly luciferase, was notably slower with JE105 70S ribosomes and 50S subunits. Further, in vitro analysis by fast kinetics revealed that single L12 dimer ribosomes from JE105 are defective in two major steps of translation, namely initiation and elongation involving translational GTPases IF2 and EF-G. Varying number of L12 dimers on the ribosome can be a mechanism in bacteria for modulating the rate of translation in response to growth condition.

  • 10.
    Zhang, Yanqing
    et al.
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Cao, Wei
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    Li, Xiaojing
    Chinese Acad Sci, Inst Microbiol, Key Lab Pathogen Microbiol & Immunol, Beijing, Peoples R China..
    Zhang, Dejiu
    Chinese Acad Sci, Inst Biophys, Key Lab RNA Biol, Beijing 100080, Peoples R China..
    Li, Ningning
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    Zhang, Yiudao
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    Zhang, Xiaoxiao
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    Qin, Yan
    Chinese Acad Sci, Inst Biophys, Key Lab RNA Biol, Beijing 100080, Peoples R China..
    Mi, Kaixia
    Chinese Acad Sci, Inst Microbiol, Key Lab Pathogen Microbiol & Immunol, Beijing, Peoples R China..
    Lei, Jianlin
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gao, Ning
    Tsinghua Univ, Sch Life Sci, Struct Biol Ctr, Key Lab Prot Sci,Minist Educ, Beijing 100084, Peoples R China..
    HflX is a ribosome-splitting factor rescuing stalled ribosomes under stress conditions2015In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 22, no 11, p. 906-913Article in journal (Refereed)
    Abstract [en]

    Adverse cellular conditions often lead to nonproductive translational stalling and arrest of ribosomes on mRNAs. Here, we used fast kinetics and cryo-EM to characterize Escherichia coil HflX, a GTPase with unknown function. Our data reveal that HflX is a heat shock-induced ribosome-splitting factor capable of dissociating vacant as well as mRNA-associated ribosomes with deacylated tRNA in the peptidyl site. Structural data demonstrate that the N-terminal effector domain of HflX binds to the peptidyl transferase center in a strikingly similar manner as that of the class I release factors and induces dramatic conformational changes in central intersubunit bridges, thus promoting subunit dissociation. Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat shock, These results suggest a primary role of HflX in rescuing translationally arrested ribosomes under stress conditions.

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