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  • 1.
    Banerjee, Debapriya
    et al.
    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.
    Protein Folding Activity of the Ribosome (PFAR): A Target for Antiprion Compounds2014In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 6, no 10, p. 3907-3924Article, review/survey (Refereed)
    Abstract [en]

    Prion diseases are fatal neurodegenerative diseases affecting mammals. Prions are misfolded amyloid aggregates of the prion protein (PrP), which form when the alpha helical, soluble form of PrP converts to an aggregation-prone, beta sheet form. Thus, prions originate as protein folding problems. The discovery of yeast prion(s) and the development of a red-/white-colony based assay facilitated safe and high-throughput screening of antiprion compounds. With this assay three antiprion compounds; 6-aminophenanthridine (6AP), guanabenz acetate (GA), and imiquimod (IQ) have been identified. Biochemical and genetic studies reveal that these compounds target ribosomal RNA (rRNA) and inhibit specifically the protein folding activity of the ribosome (PFAR). The domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active site for PFAR. 6AP and GA inhibit PFAR by competition with the protein substrates for the common binding sites on the domain V rRNA. PFAR inhibition by these antiprion compounds opens up new possibilities for understanding prion formation, propagation and the role of the ribosome therein. In this review, we summarize and analyze the correlation between PFAR and prion processes using the antiprion compounds as tools.

  • 2.
    Banerjee, Debapriya
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Vovusha, Hakkim
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Pang, Yanhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Oumata, Nassima
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Spectroscopic and DFT studies on 6-Aminophenanthridine and its derivatives provide insights in their activity towards ribosomal RNA2014In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 97, p. 194-199Article in journal (Refereed)
    Abstract [en]

    6-Aminophenanthridine (6AP), a plant alkaloid possessing antiprion activity, inhibits ribosomal RNA dependent protein folding activity of the ribosome (referred as PFAR). We have compared 6AP and its three derivatives 6AP8Cl, 6AP8CF3 and 6APi for their activity in inhibition of PFAR. Since PFAR inhibition requires 6AP and its derivatives to bind to the ribosomal RNA (rRNA), we have measured the binding affinity of these molecules to domain V of 23S rRNA using fluorescence spectroscopy. Our results show that similar to the antiprion activity, both the inhibition of PFAR and the affinity towards rRNA follow the order 6AP8CF3 > 6AP8Cl > 6AP, while 6APi is totally inactive. To have a molecular insight for the difference in activity despite similarities in structure, we have calculated the nucleus independent chemical shift using first principles density functional theory. The result suggests that the deviation of planarity in 6APi and steric hindrance from its bulky side chain are the probable reasons which prevent it from interacting with rRNA. Finally, we suggest a probable mode of action of 6AP, 6AP8CF3 and 6AP8Cl towards rRNA.

  • 3.
    Borg, Anneli
    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.
    Shiroyama, Ikue
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hauryliuk, Vasili
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Pavlov, Michael
    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.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Fusidic Acid Targets Elongation Factor G in Several Stages of Translocation on the Bacterial Ribosome2015In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 6, p. 3440-3454Article in journal (Refereed)
    Abstract [en]

    The antibiotic fusidic acid (FA) targets elongation factor G (EF-G) and inhibits ribosomal peptide elongation and ribosome recycling, but deeper mechanistic aspects of FA action have remained unknown. Using quench flow and stopped flow experiments in a biochemical system for protein synthesis and taking advantage of separate time scales for inhibited (10 s) and uninhibited (100 ms) elongation cycles, a detailed kinetic model of FA action was obtained. FA targets EF-G at an early stage in the translocation process (I), which proceeds unhindered by the presence of the drug to a later stage (II), where the ribosome stalls. Stalling may also occur at a third stage of translocation(III), just before release of EF-G from the post-translocation ribosome. We show that FA is a strong elongation inhibitor (K-50% approximate to 1 mu M), discuss the identity of the FA targeted states, and place existing cryo-EM and crystal structures in their functional context.

  • 4.
    Chai, Qian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Singh, Bhupender
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology.
    Distribution of ribosomes in action and at rest in Escherichia coli: involvement of bacterial cytoskeletonManuscript (preprint) (Other academic)
  • 5.
    Chai, Qian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Singh, Bhupender
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Metzendorf, Nicole
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dasgupta, Santanu
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Organization of Ribosomes and Nucleoids in Escherichia coli Cells during Growth and in Quiescence2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 16, p. 11342-11352Article in journal (Refereed)
    Abstract [en]

    Background: We studied ribosome and nucleoid distribution in Escherichia coli under growth and quiescence. Results: Spatially segregated ribosomes and nucleoids show drastically altered distribution in stationary phase or when treated with drugs affecting translation, transcription, nucleoid-topology, or cytoskeleton. Ribosome inheritance in daughter cells is frequently unequal. Conclusion: Cellular growth processes modulate ribosome and nucleoid distribution. Significance: This provides insight into subcellular organization of molecular machines. We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

  • 6.
    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.

  • 7.
    Chandra Sanyal, S
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Liljas, A
    The end of the beginning: structural studies of ribosomal proteins2000In: Curr Opin Struct Biol, Vol. 10, no 6, p. 633-636Article, book review (Other (popular scientific, debate etc.))
    Abstract [en]

    Work on the structural biology of ribosomes has progressed rapidly over the past few years. It has come to a stage at which the structures of the individual components are no longer of interest, except for those that still present ambiguous information ab

  • 8.
    Cordeiro, Yraima
    et al.
    Univ Fed Rio de Janeiro, Fac Pharm, BR-21941902 Rio De Janeiro, Brazil.
    Vieira, Tuane
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil.
    Kovachev, Petar Stefanov
    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.
    Silva, Jerson L.
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil.
    Modulation of p53 and prion protein aggregation by RNA2019In: Biochimica et Biophysica Acta - Proteins and Proteomics, ISSN 1570-9639, E-ISSN 1878-1454, Vol. 1867, no 10, p. 933-940Article, review/survey (Refereed)
    Abstract [en]

    Several RNA-binding proteins undergo reversible liquid-liquid phase transitions, which, in pathological conditions, might evolve into transitions to solid-state phases, giving rise to amyloid structures. Amyloidogenic and prion-like proteins, such as the tumor suppressor protein p53 and the mammalian prion protein (PrP), bind RNAs specifically or nonspecifically, resulting in changes in their propensity to undergo aggregation. Mutant p53 aggregation seems to play a crucial role in cancer through loss of function, negative dominance and gain of function. PrP conversion modulated by RNA results in highly toxic aggregates. Here, we review data on the modulatory action of RNAs on the aggregation of both proteins.

  • 9. Deroo, Stephanie
    et al.
    Hyung, Suk-Joon
    Marcoux, Julien
    Gordiyenko, Yuliya
    Koripella, Ravi Kiran
    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.
    Robinson, Carol V.
    Mechanism and Rates of Exchange of L7/L12 between Ribosomes and the Effects of Binding EF-G2012In: ACS Chemical Biology, ISSN 1554-8929, E-ISSN 1554-8937, Vol. 7, no 6, p. 1120-1127Article in journal (Refereed)
    Abstract [en]

    The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by similar to 27% and of dimers by similar to 47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.

  • 10. 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.

  • 11.
    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.

  • 12. Gao, Haixiao
    et al.
    Zhou, Zhihong
    Rawat, Urmila
    Huang, Chenhui
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Bouakaz, Lamine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Wang, Chernhoe
    Cheng, Zhihong
    Liu, Yuying
    Zavialov, Andrey
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Gursky, Richard
    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
    Song, Haiwei
    RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors2007In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 129, no 5, p. 929-941Article in journal (Refereed)
    Abstract [en]

    During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3•GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu•GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3•GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.

  • 13.
    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.

  • 14.
    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.

  • 15.
    Holm, Mikael
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Borg, Anneli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ehrenberg, Måns
    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.
    Molecular mechanism of viomycin inhibition of peptide elongation in bacteria2016In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 4, p. 978-983Article in journal (Refereed)
    Abstract [en]

    Viomycin is a tuberactinomycin antibiotic essential for treating multi-drug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of similar to 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

  • 16.
    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.

  • 17.
    Kacar, Betuel
    et al.
    NASA, Astrobiol Inst, Moffett Field, CA 94035 USA.;Harvard Univ, Organism & Evolut Biol, 26 Oxford St, Cambridge, MA 02138 USA..
    Ge, Xueliang
    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.
    Gaucher, Eric A.
    Georgia Inst Technol, Sch Biol, 950 Atlantic Dr, Atlanta, GA 30332 USA.;Georgia Inst Technol, Petit H Parker Inst Bioengn & Biosci, Atlanta, GA 30332 USA..
    Experimental Evolution of Escherichia coli Harboring an Ancient Translation Protein2017In: Journal of Molecular Evolution, ISSN 0022-2844, E-ISSN 1432-1432, Vol. 84, no 2-3, p. 69-84Article in journal (Refereed)
    Abstract [en]

    The ability to design synthetic genes and engineer biological systems at the genome scale opens new means by which to characterize phenotypic states and the responses of biological systems to perturbations. One emerging method involves inserting artificial genes into bacterial genomes and examining how the genome and its new genes adapt to each other. Here we report the development and implementation of a modified approach to this method, in which phylogenetically inferred genes are inserted into a microbial genome, and laboratory evolution is then used to examine the adaptive potential of the resulting hybrid genome. Specifically, we engineered an approximately 700-million-year-old inferred ancestral variant of tufB, an essential gene encoding elongation factor Tu, and inserted it in a modern Escherichia coli genome in place of the native tufB gene. While the ancient homolog was not lethal to the cell, it did cause a twofold decrease in organismal fitness, mainly due to reduced protein dosage. We subsequently evolved replicate hybrid bacterial populations for 2000 generations in the laboratory and examined the adaptive response via fitness assays, whole genome sequencing, proteomics, and biochemical assays. Hybrid lineages exhibit a general adaptive strategy in which the fitness cost of the ancient gene was ameliorated in part by upregulation of protein production. Our results suggest that an ancient-modern recombinant method may pave the way for the synthesis of organisms that exhibit ancient phenotypes, and that laboratory evolution of these organisms may prove useful in elucidating insights into historical adaptive processes.

  • 18.
    Koripella, Ravi Kiran
    et al.
    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.
    Peisker, Kristin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Koh, Cha San
    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.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 36, p. 30257-30267Article in journal (Refereed)
    Abstract [en]

    Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.

  • 19.
    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.

  • 20.
    Koripella, Ravi Kiran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Holm, Mikael
    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.
    Essential role of Histidine 92 in elongation factor-G in GTP hydrolysis and inorganic phosphate release during elongation of protein synthesisManuscript (preprint) (Other academic)
    Abstract [en]

    The histidine (H) residue at the apex of switch II is conserved in all translational GTPases. Thishistidine (H92) in elongation factor G (EF-G) has been implicated in GTP hydrolysis andinorganic phosphate (pi) release similar to H85 in elongation factor-Tu (EF-Tu). Mutagenesis ofH92 to alanine (A) and glutamic acid (E) showed different degrees of defect in different steps ofelongation. While H92A was ~7 times slower than wild type EF-G in ribosome mediated GTPhydrolysis, it was 100 times slower in both pi release and tRNA translocation. The H92E mutant,on the other hand, was 100 times slower in all these steps. Both mutants were significantlydefective (~1000 times slower) in tripeptide formation that which requires dissociation of EF-Gfrom the post-translocation state. Thus, our results indicate that GTP hydrolysis takes place priorto tRNA translocation, whereas Pi release occurs probably after or independent of thetranslocation step. Since translocation involves back ratcheting of the ribosomal subunits ourresults suggest that there is a cross-talk between GTP hydrolysis by EF-G and ribosomal subunitrotation. We further confirm that Pi release is essential for the next round of elongation.

  • 21.
    Korkmaz, Gürkan
    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.
    Wiens, Tobias
    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.
    Comprehensive Analysis of Stop Codon Usage in Bacteria and Its Correlation with Release Factor Abundance2014In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 289, no 44, p. 30334-30342Article in journal (Refereed)
    Abstract [en]

    We present a comprehensive analysis of stop codon usage in bacteria by analyzing over eight million coding sequences of 4684 bacterial sequences. Using a newly developed program called "stop codon counter," the frequencies of the three classical stop codons TAA, TAG, and TGA were analyzed, and a publicly available stop codon database was built. Our analysis shows that with increasing genomic GC content the frequency of the TAA codon decreases and that of the TGA codon increases in a reciprocal manner. Interestingly, the release factor 1-specific codon TAG maintains a more or less uniform frequency (similar to 20%) irrespective of the GC content. The low abundance of TAG is also valid with respect to expression level of the genes ending with different stop codons. In contrast, the highly expressed genes predominantly end with TAA, ensuring termination with either of the two release factors. Using three model bacteria with different stop codon usage (Escherichia coli, Mycobacterium smegmatis, and Bacillus subtilis), we show that the frequency of TAG and TGA codons correlates well with the relative steady state amount of mRNA and protein for release factors RF1 and RF2 during exponential growth. Furthermore, using available microarray data for gene expression, we show that in both fast growing and contrasting biofilm formation conditions, the relative level of RF1 is nicely correlated with the expression level of the genes ending with TAG.

  • 22.
    Korkmaz, Gürkan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Lind, Christoffer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Åqvist, Johan
    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.
    Characterizing an engineered release factor capable of reading all three stop codons2014In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 28, no 1, article id 569.2Article in journal (Other academic)
  • 23.
    Korkmaz, Gürkan
    et al.
    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.
    R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria Escherichia coli2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 36, p. 15134-15142Article in journal (Refereed)
    Abstract [en]

    The current understanding of the specificity of the bacterial class I release factors (RFs) in decoding stop codons has evolved beyond a simple tripeptide anticodon model. A recent molecular dynamics study for deciphering the principles for specific stop codon recognition by RFs identified Arg-213 as a crucial residue on Escherichia coli RF2 for discriminating guanine in the third position (G3). Interestingly, Arg-213 is highly conserved in RF2 and substituted by Ile-196 in the corresponding position in RF1. Another similar pair is Leu-126 in RF1 and Asp-143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of Arg-213 and Asp-143 with the corresponding RF1 residues will reduce G3 discrimination by RF2, we swapped these residues between E. coli RF1 and RF2 by site-directed mutagenesis and characterized their preference for different codons using a competitive peptide release assay. Among these, the R213I mutant of RF2 showed 5-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared with the wild type (WT). In-depth fast kinetic studies revealed that the gain in UAG reading by RF2 R213I is associated with a reduced efficiency of termination on the cognate UAA codon. Our work highlights the notion that stop codon recognition involves complex interactions with multiple residues beyond the PXT/SPF motifs. We propose that the R213I mutation in RF2 brings us one step forward toward engineering an omnipotent RF in bacteria, capable of reading all three stop codons.

  • 24.
    Kovachev, Petar Stefanov
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Rangel, Luciana Pereira
    Univ Fed Rio de Janeiro, Fac Farm, BR-21941902 Rio De Janeiro, Brazil..
    Eriksson, Jonny
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Pedrote, Murilo M
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo de Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil..
    Martins-Dinis, Mafalda Maria D C
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo de Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil..
    Edwards, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry.
    Cordeiro, Yraima
    Univ Fed Rio de Janeiro, Fac Farm, BR-21941902 Rio De Janeiro, Brazil..
    Silva, Jerson L
    Univ Fed Rio de Janeiro, Inst Bioquim Med Leopoldo de Meis, Inst Nacl Ciencia Tecnol Biol Estrutural & Bioima, BR-21941902 Rio De Janeiro, Brazil..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.2017In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 22, p. 9345-9357Article in journal (Refereed)
    Abstract [en]

    Inactivation of the tumor suppressor protein p53 by mutagenesis, chemical modification, protein-protein interaction, or aggregation has been associated with different human cancers. Although DNA is the typical substrate of p53, numerous studies have reported p53 interactions with RNA. Here, we have examined the effects of RNA of varied sequence, length, and origin on the mechanism of aggregation of the core domain of p53 (p53C) using light scattering, intrinsic fluorescence, transmission electron microscopy, thioflavin-T binding, seeding, and immunoblot assays. Our results are the first to demonstrate that RNA can modulate the aggregation of p53C and full-length p53. We found bimodal behavior of RNA in p53C aggregation. A low RNA:protein ratio (∼1:50) facilitates the accumulation of large amorphous aggregates of p53C. By contrast, at a high RNA:protein ratio (≥1:8), the amorphous aggregation of p53C is clearly suppressed. Instead, amyloid p53C oligomers are formed that can act as seeds nucleating de novo aggregation of p53C. We propose that structured RNAs prevent p53C aggregation through surface interaction and play a significant role in the regulation of the tumor suppressor protein.

  • 25. Li, Jing-Jing
    et al.
    Venkataramana, Musturi
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Sanyal, Suparna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology.
    Janson, Jan-Christer
    Surface Biotechnology. Chemistry, Department of Physical and Analytical Chemistry, Surface Biotechnology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Physical and Analytical Chemistry.
    Su, Zhi-Guo
    Immobilized β-cyclodextrin polymer coupled to agarose gel properly refolding recombinant Staphylococcus aureus elongation factor-G in combination with detergent micelle2006In: Protein Expression and Purification, Vol. 45, no 1, p. 72-79Article in journal (Refereed)
    Abstract [en]

    A novel artificial chaperone system using a combination of interactions between the unfolded protein, a detergent and a chromatographic column packed with immobilized β-cyclodextrin (β-CD) polymer coupled to an agarose gel, was introduced to refold recombinant Staphylococcus aureus elongation factor-G (EF-G). Pre-mixing of 10% Triton X-100 and unfolded EF-G at 24 mg/ml followed by a 20-fold dilution into refolding buffer led to successful capturing of EF-G by Triton X-100 resulting in formation of a detergent–protein complex at 1.2 mg/ml of final protein concentration. The complex was subsequently applied to the immobilized β-CD polymer column resulting in correct refolding of EF-G at a concentration of 530 μg/ml with 99% mass recovery. Detergent concentrations above critical micelle concentration were required for efficient capturing of EF-G at high protein concentration. Other detergents with hydrophile–lipophile-Balance values similar to that of Triton X-100 (Triton N-101, Noindet P40 (NP40), and Berol 185) also produced similar result. Soluble polymerized β-CD was more efficient than the monomer to remove the detergent from the protein complex in a batch system. Immobilized β-CD polymer column further improved the capability of detergent removal and was able to prevent aggregation that occurred with the addition of soluble β-CD polymer at high protein concentration in the batch system. The mechanism for this system-assisted refolding was tentatively interpreted: the released protein could correctly refold in an enclosed hydrophilic environment provided by the integration of matrix and β-CD polymer, and thus avoided aggregation during detergent removal.

  • 26. Li, Jing-Jing
    et al.
    Venkataramana,, Musturi
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Wang, Ai-Qing
    Sanyal, Suparna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Janson, Jan-Christer
    Chemistry, Surface Biotechnology. Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Physical and Analytical Chemistry.
    Su, Zhi-Guo
    A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G2005In: Protein Expression and Purification, Vol. 40, no 2, p. 327-335Article in journal (Refereed)
    Abstract [en]

    Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.

  • 27.
    Li, Zhifei
    et al.
    Peking Univ, Sch Life Sci, Peking Tsinghua Joint Ctr Life Sci, State Key Lab Membrane Biol, Beijing, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing, Peoples R China.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Zhang, Yixiao
    Peking Univ, Sch Life Sci, Peking Tsinghua Joint Ctr Life Sci, State Key Lab Membrane Biol, Beijing, Peoples R China.
    Zheng, Lvqin
    Peking Univ, Sch Life Sci, Peking Tsinghua Joint Ctr Life Sci, State Key Lab Membrane Biol, Beijing, Peoples R China.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gao, Ning
    Peking Univ, Sch Life Sci, Peking Tsinghua Joint Ctr Life Sci, State Key Lab Membrane Biol, Beijing, Peoples R China; Tsinghua Univ, Beijing Adv Innovat Ctr Struct Biol, Sch Life Sci, Tsinghua Peking Joint Ctr Life Sci, Beijing, Peoples R China.
    Cryo-EM structure of Mycobacterium smegmatis ribosome reveals two unidentified ribosomal proteins close to the functional centers2018In: Protein & cell, ISSN 1674-8018, Vol. 9, no 4, p. 384-388Article in journal (Other academic)
  • 28.
    Liljas, Anders
    et al.
    Lund Univ, Dept Biochem & Struct Biol, Ctr Chem & Chem Engn, Lund, Sweden.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    The enigmatic ribosomal stalk2018In: Quarterly reviews of biophysics (Print), ISSN 0033-5835, E-ISSN 1469-8994, Vol. 51, article id e12Article, review/survey (Refereed)
    Abstract [en]

    The large ribosomal subunit has a distinct feature, the stalk, extending outside the ribosome. In bacteria it is called the L12 stalk. The base of the stalk is protein uL10 to which two or three dimers of proteins bL12 bind. In archea and eukarya P1 and P2 proteins constitute the stalk. All these extending proteins, that have a high degree of flexibility due to a hinge between their N- and C-terminal parts, are essential for proper functionalization of some of the translation factors. The role of the stalk proteins has remained enigmatic for decades but is gradually approaching an understanding. In this review we summarise the knowhow about the structure and function of the ribosomal stalk till date starting from the early phase of ribosome research.

  • 29.
    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.

  • 30.
    Masuda, Isao
    et al.
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
    Igarashi, Takao
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
    Sakaguchi, Reiko
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA.;Kyoto Univ, Kyoto 6068501, Japan..
    Nitharwal, Ram G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Takase, Ryuichi
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
    Han, Kyu Young
    Univ Illinois, Dept Phys, Urbana, IL 61801 USA.;Univ Illinois, Ctr Phys Living Cells, Urbana, IL 61801 USA.;Univ Cent Florida, CREOL, Coll Opt & Photon, 4304 Scorpius St, Orlando, FL 32816 USA..
    Leslie, Benjamin J.
    Univ Illinois, Dept Phys, Urbana, IL 61801 USA.;Univ Illinois, Ctr Phys Living Cells, Urbana, IL 61801 USA.;Howard Hughes Med Inst, Baltimore, MD 21205 USA..
    Liu, Cuiping
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA.;Huazhong Agr Univ, Coll Vet Med, Wuhan, Peoples R China..
    Gamper, Howard
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
    Ha, Taekjip
    Univ Illinois, Dept Phys, Urbana, IL 61801 USA.;Univ Illinois, Ctr Phys Living Cells, Urbana, IL 61801 USA.;Howard Hughes Med Inst, Baltimore, MD 21205 USA.;Johns Hopkins Univ, Dept Biophys, Baltimore, MD 21218 USA.;Johns Hopkins Univ, Sch Med, Dept Biophys & Biophys Chem, Baltimore, MD 21205 USA.;Johns Hopkins Univ, Dept Biomed Engn, Baltimore, MD 21205 USA..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Hou, Ya-Ming
    Thomas Jefferson Univ, Dept Biochem & Mol Biol, 233 South 10th St, Philadelphia, PA 19107 USA..
    A genetically encoded fluorescent tRNA is active in live-cell protein synthesis2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 7, p. 4081-4093Article in journal (Refereed)
    Abstract [en]

    Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.

  • 31.
    Mishra, Abhishek Kumar
    et al.
    Ctr Biomed Res CBMR, Div Mol Synth & Drug Discovery, SGPGIMS Campus, Lucknow 226014, Uttar Pradesh, India.
    Morgon, Nelson Henrique
    Univ Estadual Campinas, UNICAMP, Inst Chem, Dept Phys Chem, BR-13083970 Campinas, SP, Brazil.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    de Souza, Aguinaldo Robinson
    Sao Paulo State Univ, UNESP, Sch Sci, BR-17033360 Bauru, SP, Brazil.
    Biswas, Srijit
    Ctr Biomed Res CBMR, Div Mol Synth & Drug Discovery, SGPGIMS Campus, Lucknow 226014, Uttar Pradesh, India;Univ Calcutta, Rajabazar Sci Coll Campus, Dept Chem, Kolkata 700009, W Bengal, India.
    Catalytic O- to N-Alkyl Migratory Rearrangement: Transition Metal-Free Direct and Tandem Routes to N-Alkylated Pyridones and Benzothiazolones2018In: Advanced Synthesis and Catalysis, ISSN 1615-4150, E-ISSN 1615-4169, Vol. 360, no 20, p. 3930-3939Article in journal (Refereed)
    Abstract [en]

    The present study reports the synthesis of N-alkylated pyridones and benzothiazolones via O- to N-alkyl group migration under transition metal-free TfOH-catalyzed reaction conditions for the first time, to the best of our knowledge. Primary as well as secondary alkyl groups smoothly migrate under the present reaction conditions. Moreover, a minor modification of the protocol used in this study is found to be applicable for an entirely new tandem synthesis of 2-alkoxy-N-heterocycles from the simplest starting materials in a solvent-free reaction conditions. Density Functional Theory (DFT) calculation identifies the energy species associated with the rearrangement, whereas, mechanistic experiments explore the role of the catalyst as the alkyl group transfer mediator.

  • 32.
    Mohapatra, Sonisilpa
    et al.
    Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA..
    Choi, Heejun
    Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA.;Howard Hughes Med Inst, Janelia Res Campus, Ashburn, VA USA..
    Ge, Xueliang
    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.
    Weisshaar, James C.
    Univ Wisconsin, Dept Chem, 1101 Univ Ave, Madison, WI 53706 USA..
    Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 3, article id e00300-17Article in journal (Refereed)
    Abstract [en]

    In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P-mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-P-K34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is tau(free) approximate to 16 ms. The typical residence time of an EF-P on the ribosome is very short, tau(bound) approximate to 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.

  • 33. Nguyen, Phu Hai
    et al.
    Hammoud, Hassan
    Halliez, Sophie
    Pang, Yanhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Evrard, Justine
    Schmitt, Martine
    Oumata, Nassima
    Bourguignon, Jean-Jacques
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Beringue, Vincent
    Blondel, Marc
    Bihel, Frederic
    Voisset, Cecile
    Structure-Activity Relationship Study around Guanabenz Identifies Two Derivatives Retaining Antiprion Activity but Having Lost alpha 2-Adrenergic Receptor Agonistic Activity2014In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 5, no 10, p. 1075-1082Article in journal (Refereed)
    Abstract [en]

    Guanabenz (GA) is an orally active alpha 2-adrenergic agonist that has been used for many years for the treatment of hypertension. We recently described that GA is also active against both yeast and mammalian prions in an alpha 2-adrenergic receptor-independent manner. These data suggest that this side-activity of GA could be explored for the treatment of prion-based diseases and other amyloid-based disorders. In this perspective, the potent antihypertensive activity of GA happens to be an annoying side-effect that could limit its use. In order to get rid of GA agonist activity at alpha 2-adrenergic receptors, we performed a structure-activity relationship study around GA based on changes of the chlorine positions on the benzene moiety and then on the modifications of the guanidine group. Hence, we identified the two derivatives 6 and 7 that still possess a potent antiprion activity but were totally devoid of any agonist activity at alpha 2-adrenergic receptors. Similarly to GA, 6 and 7 were also able to inhibit the protein folding activity of the ribosome (PFAR) which has been suggested to be involved in prion appearance/maintenance. Therefore, these two GA derivatives are worth being considered as drug candidates.

  • 34. Oumata, Nassima
    et al.
    Nguyen, Phu Hai
    Beringue, Vincent
    Soubigou, Flavie
    Pang, Yanhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Desban, Nathalie
    Massacrier, Catherine
    Morel, Yannis
    Paturel, Carine
    Contesse, Marie-Astrid
    Bouaziz, Serge
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Galons, Herve
    Blondel, Marc
    Voisset, Cecile
    The Toll-Like Receptor Agonist Imiquimod Is Active against Prions2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 8, p. e72112-Article in journal (Refereed)
    Abstract [en]

    Using a yeast-based assay, a previously unsuspected antiprion activity was found for imiquimod (IQ), a potent Toll-like receptor 7 (TLR7) agonist already used for clinical applications. The antiprion activity of IQ was first detected against yeast prions [PSI+] and [URE3], and then against mammalian prion both ex vivo in a cell-based assay and in vivo in a transgenic mouse model for prion diseases. In order to facilitate structure-activity relationship studies, we conducted a new synthetic pathway which provides a more efficient means of producing new IQ chemical derivatives, the activity of which was tested against both yeast and mammalian prions. The comparable antiprion activity of IQ and its chemical derivatives in the above life forms further emphasizes the conservation of prion controlling mechanisms throughout evolution. Interestingly, this study also demonstrated that the antiprion activity of IQ and IQ-derived compounds is independent from their ability to stimulate TLRs. Furthermore, we found that IQ and its active chemical derivatives inhibit the protein folding activity of the ribosome (PFAR) in vitro.

  • 35.
    Pallesen, Jesper
    et al.
    Columbia University.
    Hashem, Yaser
    Columbia University.
    Korkmaz, Gürkan
    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.
    Huang, Chenhui
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Suparna Chandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Frank, Joachim
    Columbia University.
    Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF12013In: eLife, ISSN 2050-084X, Vol. 2, p. e00411-Article in journal (Refereed)
    Abstract [en]

    Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone-RF3 bound to a non-hydrolyzable GTP-analog-have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.

  • 36.
    Pang, Yanhong
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kurella, Sriram
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Voisset, Cecile
    Samanta, Dibyendu
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Schabe, Ariane
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Das Gupta, Chanchal
    Galons, Herve
    Blondel, Marc
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The Antiprion Compound 6-Aminophenanthridine Inhibits the Protein Folding Activity of the Ribosome by Direct Competition2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 26, p. 19081-19089Article in journal (Refereed)
    Abstract [en]

    Domain V of the 23S/25S/28S rRNA of the large ribosomal subunit constitutes the active center for the protein folding activity of the ribosome (PFAR). Using in vitro transcribed domain V rRNAs from Escherichia coli and Saccharomyces cerevisiae as the folding modulators and human carbonic anhydrase as a model protein, we demonstrate that PFAR is conserved from prokaryotes to eukaryotes. It was shown previously that 6-aminophenanthridine (6AP), an antiprion compound, inhibits PFAR. Here, using UV cross-linking followed by primer extension, we show that the protein substrates and 6AP interact with a common set of nucleotides on domain V of 23S rRNA. Mutations at the interaction sites decreased PFAR and resulted in loss or change of the binding pattern for both the protein substrates and 6AP. Moreover, kinetic analysis of human carbonic anhydrase refolding showed that 6AP decreased the yield of the refolded protein but did not affect the rate of refolding. Thus, we conclude that 6AP competitively occludes the protein substrates from binding to rRNA and thereby inhibits PFAR. Finally, we propose a scheme clarifying the mechanism by which 6AP inhibits PFAR.

  • 37.
    Pavlov, Michael Y.
    et al.
    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.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Initiation of bacterial protein synthesis with wild type and novel mutants of initiation factor2011In: Ribosomes: Structure, Function and Dynamics / [ed] Marina Rodnina, Wolfgang Wintermeyer, Rachel Green, Springer-Verlag New York, 2011, p. 129-141Conference paper (Refereed)
  • 38. Qiang, Xiaoling
    et al.
    Liotta, Anthony S
    Shiloach, Joseph
    Gutierrez, J C
    Wang, Haichao
    Ochani, Mahendar
    Ochani, Kanta
    Yang, Huan
    Rabin, Aviva
    LeRoith, Derek
    Lesniak, Maxine A
    Böhm, Markus
    Maaser, Christian
    Kannengiesser, Klaus
    Donowitz, Mark
    Rabizadeh, Shervin
    Czura, Christopher J
    Tracey, Kevin J
    Westlake, Mark
    Zarfeshani, Aida
    Mehdi, Syed F
    Danoff, Ann
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Schwartz, Gary J
    Roth, Jesse
    New melanocortin-like peptide of E. coli can suppress inflammation via the mammalian melanocortin-1 receptor (MC1R): possible endocrine-like function for microbes of the gut.2017In: NPJ biofilms and microbiomes, ISSN 2055-5008, Vol. 3, article id 31Article in journal (Refereed)
    Abstract [en]

    E. coli releases a 33 amino acid peptide melanocortin-like peptide of E. coli (MECO-1) that is identical to the C-terminus of the E. coli elongation factor-G (EF-G) and has interesting similarities to two prominent mammalian melanocortin hormones, alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropin (ACTH). Note that MECO-1 lacks HFRW, the common pharmacophore of the known mammalian melanocortin peptides. MECO-1 and the two hormones were equally effective in severely blunting release of cytokines (HMGB1 and TNF) from macrophage-like cells in response to (i) endotoxin (lipopolysaccharide) or (ii) pro-inflammatory cytokine HMGB-1. The in vitro anti-inflammatoty effects of MECO-1 and of alpha-MSH were abrogated by (i) antibody against melanocortin-1 receptor (MC1R) and by (ii) agouti, an endogenous inverse agonist of MC1R. In vivo MECO-1 was even more potent than alpha-MSH in rescuing mice from death due to (i) lethal doses of LPS endotoxin or (ii) cecal ligation and puncture, models of sterile and infectious sepsis, respectively.

  • 39. Riber, Ditte
    et al.
    Venkataramana, Musturi
    Sanyal, Suparna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Duvold, Tore
    Synthesis and biological evaluation of photoaffinity labeled fusidic acid analogues.2006In: J Med Chem, ISSN 0022-2623, Vol. 49, no 5, p. 1503-5Article in journal (Refereed)
  • 40. Sörensen, Michael A
    et al.
    Elf, Johan
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Bouakaz, Elli
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Tenson, Tanel
    Sanyal, Suparna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Björk, Glenn R
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Over expression of a tRNA(Leu) isoacceptor changes charging pattern of leucine tRNAs and reveals new codon reading.2005In: J Mol Biol, ISSN 0022-2836, Vol. 354, no 1, p. 16-24Article in journal (Other scientific)
  • 41. Vestergaard, Bente
    et al.
    Sanyal, Suparna
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    Roessle, Manfred
    Mora, Liliana
    Buckingham, Richard H
    Kastrup, Jette S
    Gajhede, Michael
    Svergun, Dmitri I
    Ehrenberg, Måns
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Cell and Molecular Biology. Molekylärbiologi.
    The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure.2005In: Mol Cell, ISSN 1097-2765, Vol. 20, no 6, p. 929-38Article in journal (Other scientific)
  • 42.
    Vieler, Maximilian
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    p53 Isoforms and Their Implications in Cancer2018In: Cancers, ISSN 2072-6694, Vol. 10, no 9, article id 288Article, review/survey (Refereed)
    Abstract [en]

    In this review we focus on the major isoforms of the tumor-suppressor protein p53, dysfunction of which often leads to cancer. Mutations of the TP53 gene, particularly in the DNA binding domain, have been regarded as the main cause for p53 inactivation. However, recent reports demonstrating abundance of p53 isoforms, especially the N-terminally truncated ones, in the cancerous tissues suggest their involvement in carcinogenesis. These isoforms are Delta 40p53, Delta 133p53, and Delta 160p53 (the names indicate their respective N-terminal truncation). Due to the lack of structural and functional characterizations the modes of action of the p53 isoforms are still unclear. Owing to the deletions in the functional domains, these isoforms can either be defective in DNA binding or more susceptive to altered 'responsive elements' than p53. Furthermore, they may exert a 'dominant negative effect' or induce more aggressive cancer by the 'gain of function'. One possible mechanism of p53 inactivation can be through tetramerization with the Delta 133p53 and Delta 160p53 isoforms-both lacking part of the DNA binding domain. A recent report and unpublished data from our laboratory also suggest that these isoforms may inactivate p53 by fast aggregation-possibly due to ectopic overexpression. We further discuss the evolutionary significance of the p53 isoforms.

  • 43.
    Vovusha, Hakkim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy.
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Oumata, Nassima
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Electronic structure and spectroscopic properties of 6-aminophenanthridine and its derivatives: Insights from density functional theory2015In: International Journal of Quantum Chemistry, ISSN 0020-7608, E-ISSN 1097-461X, Vol. 115, no 13, p. 846-852Article in journal (Refereed)
    Abstract [en]

    6-Aminophenanthridine (6AP) and its derivatives show important biological activities as antiprion compounds and inhibitors of the protein folding activity of the ribosome. Both of these activities depend on the RNA binding property of these compounds, which has been recently characterized by fluorescence spectroscopy. Hence, fundamental insights into the photophysical properties of 6AP compounds are highly important to understand their biological activities. In this work, we have calculated electronic structures and optical properties of 6AP and its three derivatives 6AP8CF(3), 6AP8Cl, and 6APi by density functional theory (DFT) and time-dependent density functional theory (TDDFT). Our calculated spectra show a good agreement with the experimental absorption and fluorescence spectra, and thus, provide deep insights into the optical properties of the compounds. Furthermore, comparing the results obtained with four different hybrid functionals, we demonstrate that the accuracy of the functionals varies in the order B3LYP>PBE0>M062X>M06HF.

  • 44.
    Vovusha, Hakkim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Banerjee, Debapriya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Yadav, Manoj Kumar
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Perrozzi, Francesco
    Univ Aquila, Dept Phys & Chem Sci, Via Vetoio 10, I-67100 Laquila, Italy.
    Ottaviano, Luca
    Univ Aquila, Dept Phys & Chem Sci, Via Vetoio 10, I-67100 Laquila, Italy.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Binding Characteristics of Anticancer Drug Doxorubicin with Two-Dimensional Graphene and Graphene Oxide: Insights from Density Functional Theory Calculations and Fluorescence Spectroscopy2018In: The Journal of Physical Chemistry C, ISSN 1932-7447, E-ISSN 1932-7455, Vol. 122, no 36, p. 21031-21038Article in journal (Refereed)
    Abstract [en]

    There has been a perpetual interest in identifying suitable nano-carriers for drug delivery. In this regard, graphene-based two-dimensional materials have been proposed and demonstrated as drug carriers. In this paper, we have investigated the adsorption characteristics of a widely used anticancer drug, doxorubicin (DOX), on graphene (G) and graphene oxide (GO) by density functional theory calculations and fluorescence and X-ray photoelectron spectroscopies. From the calculated structural and electronic properties, we have concluded that G is a better binder of DOX compared to GO, which is also supported by our fluorescence measurements. The binding of DOX to G is mainly based on strong pi-pi stacking interactions. Consistent with this result, we also found that the sp(2) regions of GO interact with DOX stronger than the sp(3) regions attached with the functional groups; the binding is characterized by pi-pi and hydrogen-bonding interactions, respectively.

  • 45.
    Vovusha, Hakkim
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Sanyal, Biplab
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Theory.
    Interaction of Nucleobases and Aromatic Amino Acids with Graphene Oxide and Graphene Flakes2013In: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 4, no 21, p. 3710-3718Article in journal (Refereed)
    Abstract [en]

    In this work, we have studied interactions of nucleobases and aromatic amino acids with graphene (G) and graphene oxide (GO) flakes by ab initio density functional theory (DFT). It is evident from the results that GO complexes are stabilized by hydrogen bonding interactions whereas G complexes are stabilized by pi-pi interactions, leading to enhanced binding energies for GO complexes compared to G complexes. Moreover, time-dependent DFT (TD-DFT) calculations for the optical properties reveal that the GO nanoflakes and GO-nucleobase composite absorb visible light in the range of 400-700 nm, which may be useful for light-emitting devices. The insights obtained from our study will be useful to understand the role of GO flakes as carriers in targeted drug delivery and biosensors.

  • 46.
    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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