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  • 101.
    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.

  • 102.
    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.

  • 103.
    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.

  • 104.
    Korkmaz, Gürkan
    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 Abundance2014Data set
  • 105.
    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.

  • 106.
    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)
  • 107.
    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.

  • 108.
    Koster, Anna K.
    et al.
    Stanford Univ.
    Wood, Chase
    Stanford Univ.
    Thomas-Tran, Rhiannon
    Stanford Univ.
    Chavan, Tanmay S.
    Stanford Univ.
    Almqvist, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Choi, Kee-Hyun
    Stanford Univ; Korea Inst Sci & Technol.
    Du Bois, Justin
    Stanford Univ.
    Maduke, Merritt
    Stanford Univ.
    Developing a Novel Class of CLC Chloride-Channel Inhibitors2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 339A-339AArticle in journal (Other academic)
  • 109.
    Kovachev, Petar Stefanov
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    The role of RNA in prion aggregation and disease2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    As humanity evolved to witness an exceptionally high standard of living, Alzheimer’s, cancer and diabetes gradually replaced infections as the main limiting factors in longevity. It is both disturbing and captivating that such degenerative conditions are caused by the most ubiquitous biomolecule – the protein. Indeed, proteins are not only the most functional, but also the least understood of the cellular biopolymers. It is then not surprising that many severe human ailments are associated with aberrant proteostasis. The key, causative mechanism of proteinopathy is protein aggregation. Naturally occurring and sometimes functional, aggregation is an auxiliary pathway in protein folding. In the context of a crowded cellular environment, folding and aggregation are the least and one of the least understood molecular processes, respectively. Unravelling one can help deconstruct the other and vice versa, but also can provide mechanistic insight on degenerative proteinopathies. A special class of proteins, which appear to propagate their own aggregation, occupy center-stage in the scientific field devoted to this goal. These proteins known as prions, can exist in at least two distinct forms. With the human prion, one of those is functional and benign and the other is infectious, aggregation prone, self-replicating and fatally pathogenic. As it happens, prion disease shares many of the descriptive features of other proteinaceous neuropathies. That, and the seductive idea that prions dwell in the twilight zone between folding and aggregation, have made the prion phenomenon a fixation for many molecular biologists. This thesis, although not the product of fixation, deals with one aspect of the prion process – the involvement of a molecular cofactor.

    Of all plausible adjuvants, RNAs have been proposed as likely participants in the prion process. Their prominent secondary structures and attractive polyanionic surfaces allow RNAs to freely engage in interactions, at times transmitting conformational information through induced fit effects. The present work summarizes the influence of various RNAs on the aggregation profiles of three prionogenic model systems. The produced results indicate a generic role for RNA in the molecular processes prion propagation and aggregation. Altogether, this study illustrates a previously overlooked RNA function, of potential relevance for protein-based disease. 

    List of papers
    1. Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.
    Open this publication in new window or tab >>Distinct modulatory role of RNA in the aggregation of the tumor suppressor protein p53 core domain.
    Show others...
    2017 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 22, p. 9345-9357Article in journal (Refereed) Published
    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.

    Place, publisher, year, edition, pages
    American Society for Biochemistry and Molecular Biology, 2017
    Keywords
    RNA, amyloid, domain V of 23S rRNA, fluorescence, kinetics, p53, p53C, prion, protein aggregation, protein folding
    National Category
    Cell Biology
    Identifiers
    urn:nbn:se:uu:diva-327976 (URN)10.1074/jbc.M116.762096 (DOI)000402538900028 ()28420731 (PubMedID)
    Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2018-01-14
    2. Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregation
    Open this publication in new window or tab >>Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregation
    Show others...
    (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351XArticle in journal (Other academic) Submitted
    Abstract [en]

    Whether nucleic acids act as cofactors in the aggregation of prion proteins is still under debate. By employing RNAs of various source and size, we have studied the role of RNA in the aggregation of murine recombinant prion protein (rPrP23-231) using Rayleigh light scattering, dynamic light scattering, sedimentation, transmission electron microscopy, circular dichroism and isothermal titration calorimetry. We find that RNA modulates rPrP23-231 aggregation in a concentration dependent manner, affecting both the extent and rate of the process; the latter evident from fast kinetics measurements of rPrP23-231 aggregation using stopped-flow technique. At lower concentration, RNA stimulates amorphous aggregation of rPrP23-231, and at higher concentration it, instead, facilitates formation of oligomeric species capable of seeding de novo aggregation of rPrP23-231. Furthermore, RNA co-sediments with rPrP23-231. This leads to partial RNase resistance of RNA and secondary structure alterations in the protein, indicating a direct interaction between the two. Sequence analysis of the RNA co-aggregated with rPrP23-231 suggests that the interaction is not specific to RNA sequence. Alternatively, rPrP23-231 interaction with RNA appears site-specific and mediated by the N-terminus. Our study demonstrates the effective modulation of rPrP23-231 aggregation by RNA and puts forward the idea of the potential role of RNA in protein aggregation as a whole.

    Keywords
    prion, RNA, cofactor, RNA-protein interaction, aggregation, kinetics
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Molecular Cellbiology
    Identifiers
    urn:nbn:se:uu:diva-338856 (URN)
    Available from: 2018-01-14 Created: 2018-01-14 Last updated: 2018-01-14
    3. Intervention of ribosomal RNA in HET-s prion aggregation Intervention of ribosomal RNA in HET-s prion aggregation
    Open this publication in new window or tab >>Intervention of ribosomal RNA in HET-s prion aggregation Intervention of ribosomal RNA in HET-s prion aggregation
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The role of nucleic acids in prion aggregation / disaggregation has remained unclear. Here, using HET-s prion from Podospora anserina as a model system, we have studied the role of RNA, particularly different domains of ribosomal RNA, in its aggregation process. Our results show that domain V rRNA, from the large subunit of the ribosome, substantially prevents amyloid aggregation of the HET-s prion in a concentration dependent manner. Instead, it promotes the formation of oligomeric seeds, which facilitate de novo HET-s aggregation. The interaction sites for the HET-s prion on domain V rRNA were also identified and shown to overlap with the sites previously found to responsible for the protein folding activity of the ribosome (PFAR). This study provides a missing link between the role of rRNA-based PFAR and prion propagation.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Molecular Cellbiology
    Identifiers
    urn:nbn:se:uu:diva-338605 (URN)
    Available from: 2018-01-14 Created: 2018-01-14 Last updated: 2018-01-14
  • 110.
    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.

  • 111.
    Kovachev, Petar Stefanov
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gomes, Mariana P. B.
    Instituto de Tecnologia em Imunobiológicos, Bio-Manguinhos, FIOCRUZ, 21040-900, Brazil.
    Cordeiro, Yraima
    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Prosdocimi, Francisco
    Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de Ciência Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Ferreira, Natália C.
    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Valadão, Leticia P. Felix
    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Macedo, Bruno
    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Fernandes, Patricia N.
    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Rangel, Luciana P.
    Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Silva, Jerson L.
    Instituto de Bioquímica Médica Leopoldo de Meis, Instituto Nacional de Ciência Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-902, Brazil.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Direct involvement of RNA in mammalian prion protein aggregation: Involvement of RNA in rPrP aggregationIn: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351XArticle in journal (Other academic)
    Abstract [en]

    Whether nucleic acids act as cofactors in the aggregation of prion proteins is still under debate. By employing RNAs of various source and size, we have studied the role of RNA in the aggregation of murine recombinant prion protein (rPrP23-231) using Rayleigh light scattering, dynamic light scattering, sedimentation, transmission electron microscopy, circular dichroism and isothermal titration calorimetry. We find that RNA modulates rPrP23-231 aggregation in a concentration dependent manner, affecting both the extent and rate of the process; the latter evident from fast kinetics measurements of rPrP23-231 aggregation using stopped-flow technique. At lower concentration, RNA stimulates amorphous aggregation of rPrP23-231, and at higher concentration it, instead, facilitates formation of oligomeric species capable of seeding de novo aggregation of rPrP23-231. Furthermore, RNA co-sediments with rPrP23-231. This leads to partial RNase resistance of RNA and secondary structure alterations in the protein, indicating a direct interaction between the two. Sequence analysis of the RNA co-aggregated with rPrP23-231 suggests that the interaction is not specific to RNA sequence. Alternatively, rPrP23-231 interaction with RNA appears site-specific and mediated by the N-terminus. Our study demonstrates the effective modulation of rPrP23-231 aggregation by RNA and puts forward the idea of the potential role of RNA in protein aggregation as a whole.

  • 112. Kronqvist, Nina
    et al.
    Otikovs, Martins
    Chmyrov, Volodymyr
    Chen, Gefei
    Andersson, Marlene
    Nordling, Kerstin
    Landreh, Michael
    Sarr, Medoune
    Jornvall, Hans
    Wennmalm, Stefan
    Widengren, Jerker
    Meng, Qing
    Rising, Anna
    Otzen, Daniel
    Knight, Stefan D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Jaudzems, Kristaps
    Johansson, Jan
    Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 3254-Article in journal (Refereed)
    Abstract [en]

    The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

  • 113.
    Krüger, Dennis M.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Micelle Maker: An Online Tool for Generating Equilibrated Micelles as Direct Input for Molecular Dynamics Simulations2017In: ACS OMEGA, ISSN 2470-1343, Vol. 2, no 8, p. 4524-4530Article in journal (Refereed)
    Abstract [en]

    Micelles play an important role in both experimental and computational studies of the effect of lipid interactions on biological systems. The spherical geometry and the dynamical behavior of micelles makes generating micelle structures for use in molecular simulations challenging. An easy tool for generating simulation-ready micelle models, covering a broad range of lipids, is highly desirable. Here, we present a new Web server, Micelle Maker, which can provide equilibrated micelle models as a direct input for subsequent molecular dynamics simulations from a broad range of lipids (currently 25 lipid types, including 24 glycolipids). The Web server, which is available at http:// www. micellemaker. net, uses error checking routines to prevent clashes during the initial placement of the lipids and uses AMBER's GLYCAM library for generating minimized or equilibrated micelle models, but the resulting structures can be used as starting points for simulations with any force field or simulation package. Extensive validation simulations with an overall simulation time of 12 mu s using eight micelle models where assembly information is available show that all of the micelles remain very stable over the whole simulation time. Finally, we discuss the advantages of Micelle Maker relative to other approaches in the field.

  • 114.
    Kulkarni, Yashraj S.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Liao, Qinghua
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Petrovic, Dusan
    Forschungszentrum Julich, Inst Complex Syst Struct Biochem, D-52425 Julich, Germany..
    Krüger, Dennis M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Strodel, Birgit
    Forschungszentrum Julich, Inst Complex Syst Struct Biochem, D-52425 Julich, Germany.;Heinrich Heine Univ Dusseldorf, Inst Theoret & Computat Chem, Univ Str 1, D-40225 Dusseldorf, Germany..
    Amyes, Tina L.
    SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA..
    Richard, John P.
    SUNY Buffalo, Dept Chem, Buffalo, NY 14260 USA..
    Kamerlin, Shina C. L.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase2017In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 30, p. 10514-10525Article in journal (Refereed)
    Abstract [en]

    Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

  • 115.
    Kwiatkowski, Marek
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wang, Jinfan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA2014In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 25, no 11, p. 2086-2091Article in journal (Refereed)
    Abstract [en]

    Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minus) CA is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minus) CA transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.

  • 116.
    Larsson, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Virus Capsid Dissolution Studied by Microsecond Molecular Dynamics Simulations2012In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 8, no 5, p. e1002502-Article in journal (Refereed)
    Abstract [en]

    Dissolution of many plant viruses is thought to start with swelling of the capsid caused by calcium removal following infection, but no high-resolution structures of swollen capsids exist. Here we have used microsecond all-atom molecular simulations to describe the dynamics of the capsid of satellite tobacco necrosis virus with and without the 92 structural calcium ions. The capsid expanded 2.5% upon removal of the calcium, in good agreement with experimental estimates. The water permeability of the native capsid was similar to that of a phospholipid membrane, but the permeability increased 10-fold after removing the calcium, predominantly between the 2-fold and 3-fold related subunits. The two calcium binding sites close to the icosahedral 3-fold symmetry axis were pivotal in the expansion and capsid-opening process, while the binding site on the 5-fold axis changed little structurally. These findings suggest that the dissociation of the capsid is initiated at the 3-fold axis.

  • 117. Liao, Qinghua
    et al.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Strodel, Birgit
    Development and Application of a Nonbonded Cu2+ Model That Includes the Jahn-Teller Effect2015In: Journal of Physical Chemistry Letters, ISSN 1948-7185, E-ISSN 1948-7185, Vol. 6, no 13, p. 2657-2662Article in journal (Refereed)
    Abstract [en]

    Metal ions are both ubiquitous to and crucial in biology. In classical simulations, they are typically described as simple van der Waals spheres, making it difficult to provide reliable force field descriptions for them. An alternative is given by nonbonded dummy models, in which the central metal atom is surrounded by dummy particles that each carry a partial charge. While such dummy models already exist for other metal ions, none is available yet for Cu2+ because of the challenge to reproduce the Jahn-Teller distortion. This challenge is addressed in the current study, where, for the first time, a dummy model including a Jahn-Teller effect is developed for Cu2+. We successfully validate its usefulness by studying metal binding in two biological systems: the amyloid-beta peptide and the mixed-metal enzyme superoxide dismutase. We believe that our parameters will be of significant value for the computational study of Cu2+'-dependent biological systems using classical models.

  • 118. Liljas, Anders
    et al.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Structural aspects of protein synthesis2013 (ed. 2nd)Book (Other academic)
  • 119.
    Liljas, Anders
    et al.
    Department of Biochemistry and Structural Biology, Lund University.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Comment on "The mechanism for activation of GTP hydrolysis on the ribosome"2011In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 333, no 6038, p. 37-Article in journal (Refereed)
    Abstract [en]

    Voorhees et al. (Reports, 5 November 2010, p. 835) determined the structure of elongation factor Tu (EF-Tu) and aminoacyl–transfer RNA bound to the ribosome with a guanosine triphosphate (GTP) analog. However, their identification of histidine-84 of EF-Tu as deprotonating the catalytic water molecule is problematic in relation to their atomic structure; the terminal phosphate of GTP is more likely to be the proper proton acceptor.

  • 120.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gullberg, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Synthetic Biology: A Lab Manual2014 (ed. 1st)Book (Refereed)
  • 121. Lu, Xuedong
    et al.
    Nie, Shuping
    Xia, Chengjing
    Huang, Lie
    He, Ying
    Wu, Runxiang
    Zhang, Li
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    A rapid two-step algorithm detects and identifies clinical macrolide and beta-lactam antibiotic resistance in clinical bacterial isolates2014In: Journal of Microbiological Methods, ISSN 0167-7012, E-ISSN 1872-8359, Vol. 102, p. 26-31Article in journal (Refereed)
    Abstract [en]

    Purpose: Aiming to identify macrolide and beta-lactam resistance in clinical bacterial isolates rapidly and accurately, a two-step algorithm was developed based on detection of eight antibiotic resistance genes. Methods: Targeting at genes linked to bacterial macrolide (msrA, ermA, ermB, and ermC) and beta-lactam (bla(TEM), bla(SHV), bla(CTX-N-1), bin(CTX-M-9)) antibiotic resistances, this method includes a multiplex real-time PCR, a melting temperature profile analysis as well as a liquid bead microarray assay. Liquid bead microarray assay is applied only when indistinguishable T-m profile is observed. Results: The clinical validity of this method was assessed on clinical bacterial isolates. Among the total 580 isolates that were determined by our diagnostic method, 75% of them were identified by the multiplex real-time PCR with melting temperature analysis alone, while the remaining 25% required both multiplex real-time PCR with melting temperature analysis and liquid bead microarray assay for identification. Compared with the traditional phenotypic antibiotic susceptibility test, an overall agreement of 81.2% (kappa = 0.614, 95% Cl = 0550-0.679) was observed, with a sensitivity and specificity of 87.7% and 73% respectively. Besides, the average test turnaround time is 3.9 h, which is much shorter in comparison with more than 24 h for the traditional phenotypic tests. Conclusions: Having the advantages of the shorter operating time and comparable high sensitivity and specificity with the traditional phenotypic test, our two-step algorithm provides an efficient tool for rapid determination of macrolide and beta-lactam antibiotic resistances in clinical bacterial isolates.  

  • 122.
    Ma, Huan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Szeler, Klaudia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Linking coupled motions and entropic effects to the catalytic activity of 2-deoxyribose-5-phosphate aldolase (DERA)2016In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 7, p. 1415-1421Article in journal (Refereed)
    Abstract [en]

    DERA, 2-deoxyribose-5-phosphate aldolase, catalyzes the retro-aldol cleavage of 2-deoxy-ribose-5-phosphate (dR5P) into glyceraldehyde-3-phosphate (G3P) and acetaldehyde in a branch of the pentose phosphate pathway. In addition to the physiological reaction, DERA also catalyzes the reverse addition reaction and, hence, is an interesting candidate for biocatalysis of carboligation reactions, which are central to synthetic chemistry. An obstacle to overcome for this enzyme to become a truly useful biocatalyst, however, is to relax the very strict dependency of this enzyme on phoshorylated substrates. We have studied herein the role of the non-canonical phosphate-binding site of this enzyme, consisting of Ser238 and Ser239, by site-directed and site-saturation mutagenesis, coupled to kinetic analysis of mutants. In addition, we have performed molecular dynamics simulations on the wild-type and four mutant enzymes, to analyse how mutations at this phosphate-binding site may affect the protein structure and dynamics. Further examination of the S239P mutant revealed that this variant increases the enthalpy change at the transition state, relative to the wild-type enzyme, but concomitant loss in entropy causes an overall relative loss in the TS free energy change. This entropy loss, as measured by the temperature dependence of catalysed rates, was mirrored in both a drastic loss in dynamics of the enzyme, which contributes to phosphate binding, as well as an overall loss in anti-correlated motions distributed over the entire protein. Our combined data suggests that the degree of anticorrelated motions within the DERA structure is coupled to catalytic efficiency in the DERA-catalyzed retro-aldol cleavage reaction, and can be manipulated for engineering purposes.

  • 123.
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Ribosomal Stalk Protein L12: Structure, Function and Application2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ribosomal stalk proteins are known to play important role in protein synthesis. The ‘stalk’, an extended structure on the large subunit of the ribosome is composed mainly of two to three dimers of L12 and one L10 protein, which forms the base of the stalk. In E. coli, four copies of L12 molecules exist as dimer of dimers forming the pentameric L8 complex together with L10. This thesis is a collection of four interlinked studies on the structure, function and application of the ribosomal stalk protein L12. In the first study, we have mapped the interaction sites of the four major translation GTPase factors (IF2, EF-Tu, EF-G & RF3) on L12 molecule using heteronuclear NMR spectroscopy. Surprisingly, all these factors produced an overlapping interaction map spanning two α-helices on the C terminal domain of L12, thereby suggesting a general nature of the interaction between L12 and the GTPase factors. L12 is known to stimulate GTPase activity of the elongation factors EF-Tu and EF-G. Here, we have clarified the role of L12 in IF2 mediated initiation of protein synthesis. Our data suggest that rapid subunit association requires a specific interaction between the L12 protein on the 50S and IF2·GTP on the 30S preinitiation complex. We have also shown that L12 is not a GAP for IF2 and GTP hydrolysis triggers IF2 release from the 70S initiation complex. The next question we have addressed is why multiple copies of L12 dimer are needed on the ribosome. For this purpose, we created a pure E. coli strain JE105, where the terminal part of rplJ gene coding for the binding site of one L12 dimer on protein L10 was deleted in the chromosomal locus. Using ribosomes with single L12 dimer we have observed that the rate of the initiation and elongation involving IF2 and EF-G gets most compromised, which in turn decreases the growth rate of the bacteria.  This study also indicates that L12 can interact with different GTPase factors in a specialized manner. Lastly, we have developed an application making advantage of the multiple L12 dimers on the ribosome. By inserting a (His)6-tag at the C-terminus of the L12 protein we have created a novel E. coli strain (JE28), where all ribosomes are tetra-(His)6-tagged. Further, we have developed a single step method for purification of the active (His)6-tagged ribosomes from JE28.

    List of papers
    1. The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain
    Open this publication in new window or tab >>The ribosomal stalk binds to translation factors IF2, EF-Tu, EF-G and RF3 via a conserved region of the L12 C-terminal domain
    Show others...
    2007 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 365, no 2, p. 468-479Article in journal (Refereed) Published
    Abstract [en]

    Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each of which catalyzes a major step of translation in a GTP-dependent fashion. Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which forms a flexible "stalk" on the ribosome. Using heteronuclear NMR spectroscopy we demonstrate that L12 binds directly to the factors IF2, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 involved in these interactions. Factor-dependent chemical shift changes show that all four factors bind to the same region of the C-terminal domain of L12. This region includes three strictly conserved residues, K70, L80, and E82, and a set of highly conserved residues, including V66, A67, V68 and G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor-dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12.

    Keywords
    L12, ribosome, GTPase, NMR spectroscopy, protein synthesis
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-146545 (URN)10.1016/j.jmb.2006.10.025 (DOI)000243243200015 ()17070545 (PubMedID)
    Available from: 2011-02-17 Created: 2011-02-17 Last updated: 2017-12-11Bibliographically approved
    2. The Ribosomal Stalk Plays a Key Role in IF2-Mediated Association of the Ribosomal Subunits
    Open this publication in new window or tab >>The Ribosomal Stalk Plays a Key Role in IF2-Mediated Association of the Ribosomal Subunits
    2010 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 399, no 1, p. 145-153Article in journal (Refereed) Published
    Abstract [en]

    Ribosomal "stalk" protein L12 is known to activate translational GTPases EF-G and EF-Tu, but not much is known about its role in relation to other two translational G factors, IF2 and RF3. Here, we have clarified the role of L12 in IF2-mediated initiation of bacterial protein synthesis. With fast kinetics measurements, we have compared L12-depleted 50S subunits with the native ones in subunit association, GTP hydrolysis, Pi (inorganic phosphate) release and IF2 release assays. L12 depletion from 50S subunit slows the subunit association step significantly (similar to 40 fold) only when IF2.GTP is present on the 30S preinitiation complex. This demonstrates that rapid subunit association depends on a specific interaction between the L12 stalk on the 50S subunit and IF2.GTP on the 30S subunit. L12 depletion, however, did not affect the individual rates of the subsequent steps including GTP hydrolysis on IF2 and Pi release. Thus, L12 is not a GTPase activating protein (GAP) for IF2 unlike as suggested for EF-G and EF-Tu.

    Keywords
    ribosome, translation initiation, L12, IF2, subunit association
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-136263 (URN)10.1016/j.jmb.2010.04.009 (DOI)000278779900012 ()
    Available from: 2010-12-11 Created: 2010-12-11 Last updated: 2017-12-11Bibliographically approved
    3. Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G
    Open this publication in new window or tab >>Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G
    Show others...
    2012 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, no 5, p. 2054-2064Article in journal (Refereed) Published
    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.

    Keywords
    ribosome, translation initiation, L12, IF2, subunit association, protein synthesis
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biology with specialization in Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-157694 (URN)10.1093/nar/gkr1031 (DOI)000302019900022 ()22102582 (PubMedID)
    Available from: 2012-03-13 Created: 2011-08-22 Last updated: 2017-12-08Bibliographically approved
    4. A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
    Open this publication in new window or tab >>A single-step method for purification of active His-tagged ribosomes from a genetically engineered Escherichia coli
    2009 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 37, no 2, p. e15-Article in journal (Refereed) Published
    Abstract [en]

    With the rapid development of the ribosome field in recent years a quick, simple and high-throughput method for purification of the bacterial ribosome is in demand. We have designed a new strain of Escherichia coli (JE28) by an in-frame fusion of a nucleotide sequence encoding a hexa-histidine affinity tag at the 3-end of the single copy rplL gene (encoding the ribosomal protein L12) at the chromosomal site of the wild-type strain MG1655. As a result, JE28 produces a homogeneous population of ribosomes (His)(6)-tagged at the C-termini of all four L12 proteins. Furthermore, we have developed a single-step, high-throughput method for purification of tetra-(His)(6)-tagged 70S ribosomes from this strain using affinity chromatography. These ribosomes, when compared with the conventionally purified ones in sucrose gradient centrifugation, 2D-gel, dipeptide formation and a full-length protein synthesis assay showed higher yield and activity. We further describe how this method can be adapted for purification of ribosomal subunits and mutant ribosomes. These methodologies could, in principle, also be used to purify any functional multimeric complex from the bacterial cell.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-137336 (URN)10.1093/nar/gkn992 (DOI)000262963400006 ()
    Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2017-12-11Bibliographically approved
  • 124.
    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.

  • 125.
    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.

  • 126.
    Maximoff, Sergey N.
    et al.
    Loyola Univ, Dept Chem & Biochem, 1032 W Sheridan Rd, Chicago, IL 60660 USA..
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Florian, Jan
    Loyola Univ, Dept Chem & Biochem, 1032 W Sheridan Rd, Chicago, IL 60660 USA..
    DNA Polymerase lambda Active Site Favors a Mutagenic Mispair between the Enol Form of Deoxyguanosine Triphosphate Substrate and the Keto Form of Thymidine Template: A Free Energy Perturbation Study2017In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 121, no 33, p. 7813-7822Article in journal (Refereed)
    Abstract [en]

    Human DNA polymerise lambda is an intermediate fidelity member of the X family, which plays a role in DNA repair. Recent X-ray diffraction structures of a ternary complex of a loop-deletion mutant of polymerase 2, a deoxyguanosine triphosphate analogue, and a gapped DNA show that guanine and thymine form a mutagenic mispair with an . unexpected Watson Crick-like geometry rather than a wobble geometry. Hence, there is an intriguing possibility that either thyMine in the DNA or guanine in the deoxyguanosine triphosphate analogue may Spend, a substantial fraction of time in a deprotonated or enol form (both are minor species in aqueous solution) in the active site of the,polymerase lambda mutant. The experiments do not determine particular forms of the nucleobases that contribute to this mutagenic mispair. Thus, We investigate the thermodynamics of formation of various mispairs between guanine and thymine in the ternary complex at a neutral pH using classical molecular dynamics simulations and the free energy perturbation method. Our free energy calculations, as well as a comparison of the experimental and computed structures of mispairs, indicate that the Watson-Crick-like mispair between the enol tautomer of guanine and the keto tautomer of thymine is dominant. The wobble mispair between the keto forms of guanine and thymine and the Watson Crick-like mispair between the keto tautomer of guanine and the enol tautomer of thymine are less prevalent, and mispairs that involve deprotonated guanine or thymine are thermodynamically unlikely. These findings are consistent with the experiment and relevant for understanding mechanisms of spontaneous mutagenesis.

  • 127.
    Mellenius, Harriet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Exploring and predicting DNA template dependent variation in transcription2012Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Reliable transmission of information from DNA to proteins is a pre-requisite for all life, where substitution errors in the polypeptide chain may arise from transcription, aminoacylation of tRNAs or translation. The fidelity control mechanisms in transcription have nevertheless received little attention, based on the assumption that the transcriptional error is masked by the translational error. This thesis shows how accuracy theory can be applied to transcription to elucidate the principles of transcriptional accuracy. The DNA template dependent transcriptional accuracy variation is studied through modelling based on transition state theory, using thermodynamic properties of the nucleic acids in the transcription bubble. The models show that the error frequency variation in transcription causes it to surpass the translational error in some sequence contexts, making transcription a significant source of amino acids substitution errors.

    List of papers
    1. Large DNA Template Dependent Error Variation During Transcription
    Open this publication in new window or tab >>Large DNA Template Dependent Error Variation During Transcription
    2014 (English)In: Biophysics and Structure to Counter Threats and Challenges / [ed] Joseph D. Puglisi, Manolia V. Margaris, Springer Netherlands, 2014, , p. 19p. 39-57Chapter in book (Other academic)
    Abstract [en]

    The accuracy of an enzymatic reaction system is the propensity toprocess the correct, or cognate, substrate over similar, non-cognate substrates. Thisis of particular importance to polymerization reactions with a template sequence,like transcription, translation and replication. A theoretical framework for theanalysis of accuracy control is presented, including initial substrate selection andkinetic proofreading. This framework allows for analysis not only of the efficiencyof accuracy control, but also its source in standard free energy differences andequilibrium constants and its relation to the rate of product formation. A key featureis the separation of the selection in a context dependent discard parameter anda context independent discrimination parameter. When the theory is applied tothe example of prokaryote transcription, it is shown that the discard parameter,composed by experimentally well-defined values, induces a large template sequencedependent error rate variation.

    Place, publisher, year, edition, pages
    Springer Netherlands, 2014. p. 19
    Series
    NATO Science for Peace and Security Series B: Physics and Biophysics, ISSN 1874-6500
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biology with specialization in Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-181002 (URN)10.1007/978-94-007-4923-8_3 (DOI)978-94-007-4922-1 (ISBN)
    Conference
    Biophysics and Structure to Counter Threats and Challenges
    Funder
    VINNOVA
    Available from: 2012-09-14 Created: 2012-09-14 Last updated: 2015-10-20
    2. Large transcriptional accuracy variation advocate transcription as a source of amino acid sequence errors
    Open this publication in new window or tab >>Large transcriptional accuracy variation advocate transcription as a source of amino acid sequence errors
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biology with specialization in Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-181003 (URN)
    Funder
    Vinnova
    Available from: 2012-09-14 Created: 2012-09-14 Last updated: 2012-10-02
  • 128.
    Mellenius, Harriet
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Speed and accuracy in transcription and translation: Modelling of transcript and polypeptide elongation2015Doctoral thesis, comprehensive summary (Other academic)
    List of papers
    1. Large DNA Template Dependent Error Variation During Transcription
    Open this publication in new window or tab >>Large DNA Template Dependent Error Variation During Transcription
    2014 (English)In: Biophysics and Structure to Counter Threats and Challenges / [ed] Joseph D. Puglisi, Manolia V. Margaris, Springer Netherlands, 2014, , p. 19p. 39-57Chapter in book (Other academic)
    Abstract [en]

    The accuracy of an enzymatic reaction system is the propensity toprocess the correct, or cognate, substrate over similar, non-cognate substrates. Thisis of particular importance to polymerization reactions with a template sequence,like transcription, translation and replication. A theoretical framework for theanalysis of accuracy control is presented, including initial substrate selection andkinetic proofreading. This framework allows for analysis not only of the efficiencyof accuracy control, but also its source in standard free energy differences andequilibrium constants and its relation to the rate of product formation. A key featureis the separation of the selection in a context dependent discard parameter anda context independent discrimination parameter. When the theory is applied tothe example of prokaryote transcription, it is shown that the discard parameter,composed by experimentally well-defined values, induces a large template sequencedependent error rate variation.

    Place, publisher, year, edition, pages
    Springer Netherlands, 2014. p. 19
    Series
    NATO Science for Peace and Security Series B: Physics and Biophysics, ISSN 1874-6500
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biology with specialization in Molecular Biotechnology
    Identifiers
    urn:nbn:se:uu:diva-181002 (URN)10.1007/978-94-007-4923-8_3 (DOI)978-94-007-4922-1 (ISBN)
    Conference
    Biophysics and Structure to Counter Threats and Challenges
    Funder
    VINNOVA
    Available from: 2012-09-14 Created: 2012-09-14 Last updated: 2015-10-20
    2. Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E-coli rrn Operons
    Open this publication in new window or tab >>Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E-coli rrn Operons
    2014 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 106, no 1, p. 55-64Article in journal (Refereed) Published
    Abstract [en]

    Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation.

    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-217653 (URN)10.1016/j.bpj.2013.11.4487 (DOI)000329407700010 ()
    Available from: 2014-02-12 Created: 2014-02-04 Last updated: 2017-12-06Bibliographically approved
    3. DNA Template Dependent Accuracy Variation of Nucleotide Selection in Transcription
    Open this publication in new window or tab >>DNA Template Dependent Accuracy Variation of Nucleotide Selection in Transcription
    2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, article id e0119588Article in journal (Refereed) Published
    Abstract [en]

    It has been commonly assumed that the effect of erroneous transcription of DNA genes into messenger RNAs on peptide sequence errors are masked by much more frequent errors of mRNA translation to protein. We present a theoretical model of transcriptional accuracy. It uses experimentally estimated standard free energies of double-stranded DNA and RNA/DNA hybrids and predicts a DNA template dependent transcriptional accuracy variation spanning several orders of magnitude. The model also identifies high-error as well a high-accuracy transcription motifs. The source of the large accuracy span is the context dependent variation of the stacking free energy of pairs of correct and incorrect base pairs in the ever moving transcription bubble. Our model predictions have direct experimental support from recent single molecule based identifications of transcriptional errors in the C. elegans transcriptome. Our conclusions challenge the general view that amino acid substitution errors in proteins are mainly caused by translational errors. It suggests instead that transcriptional error hotspots are the dominating source of peptide sequence errors in some DNA template contexts, while mRNA translation is the major cause of protein errors in other contexts.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-252228 (URN)10.1371/journal.pone.0119588 (DOI)000351987300062 ()25799551 (PubMedID)
    Funder
    VINNOVAWenner-Gren FoundationsKnut and Alice Wallenberg Foundation
    Available from: 2015-05-06 Created: 2015-05-04 Last updated: 2017-12-04Bibliographically approved