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  • 1.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Mutants of EF-Tu defective in binding aminoacyl-tRNA1996In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 382, no 3, p. 297-303Article in journal (Refereed)
    Abstract [en]

    Five single amino acid substitution variants of EF-Tu from Salmonella typhimurium were tested for their ability to promote poly(U)-translation in vitro. The substitutions are Leu120Gln, Gln124Arg and Tyr160 (Asp or Asn or Cys). They were selected by their kirromycin resistant phenotypes and all substitutions are in domain I at the interface between domains I and III of the EF-Tu · GTP configuration. The different EF-Tu variants exhibit a spectrum of phenotypes. First, k(cat)/K(M) for the interaction between ternary complex and the programmed ribosome is apparently reduced by the substitutions Leu120Gln, Gln124Arg and Tyr160Cys. Second, this reduction is caused by a defect in the interaction between these EF-Tu variants and aminoacyl-tRNA during translation. Third, in four cases out of five the affinity of the complex between EF-Tu · GTP and aminoacyl-tRNA is significantly decreased. The most drastic reduction is observed for the Gln124Arg change, where the association constant is 30-fold lower than in the mild-type case. Fourth, missense errors are increased as well as decreased by the different amino acid substitutions. Finally, the dissociation rate constant (k(d)) for the release of GDP from EF-Tu is increased 6-fold by the Tyr160Cys substitution, but remains unchanged in the four other cases. These results show that the formation of ternary complex is sensitive to many different alterations in the domain I-III interface of EF-Tu.

  • 2.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Homologous recombination between the tuf genes of Salmonella typhimurium1996In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 260, no 4, p. 506-522Article in journal (Refereed)
    Abstract [en]

    The genes coding for the translation factor EF-Tu, tufA and tufB are separated by over 700 kb on the circular chromosome of Salmonella typhimurium. The coding regions of these genes have 99% identity at the nucleotide level in spite of the presumed ancient origin of the gene duplication. Sequence comparisons between S. typhimurium and Escherichiacoli suggest that within each species the two tuf genes are evolving inconcert. Here we show that each of the S. typhimurium tuf genes cantransfer genetic information to the other. In our genetic system thetransfers are seen as non-reciprocal, i.e. as gene conversion events.However, the mechanism of recombination could be reciprocal, with sisterchromosome segregation and selection leading to the isolation of aparticular class of recombinant. The amount of sequence informationtransferred in individual recombination events varies, but can be close tothe entire length of the gene. The recombination is RecABCD-dependent,and is opposed by MutSHLU mismatch repair. In the wild-type, this typeof recombination occurs at a rate that is two or three orders of magnitudegreater than the nucleotide substitution rate. The rate of recombinationdiffers by six orders of magnitude between a recA and a mutS strain.Mismatch repair reduces the rate of this recombination 1000-fold. The rateof recombination also differs by one order of magnitude depending onwhich tuf gene is donating the sequence selected for. We discuss threeclasses of model that could, in principle, account for the sequencetransfers: (1) tuf mRNA mediated recombination; (2) non-allelic reciprocalrecombination involving sister chromosomes; (3) non-allelic geneconversion involving sister chromosomes, initiated by a double-strandbreak close to one tuf gene. Although the mechanism remains to bedetermined, the effect on the bacterial cells is tuf gene sequencehomogenisation. This recombination phenomenon can account for theconcerted evolution of the tuf genes.

  • 3.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Liljas, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Mutations to kirromycin resistance occur in the interface of domains I and III of EF-Tu.GTP1994In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 352, p. 118-122Article in journal (Refereed)
    Abstract [en]

    The antibiotic kirromycin inhibits protein synthesis by binding to EF-Tu and preventing its release from the ribosome after GTP hydrolysis.We have isolated and sequenced a collection of kirromycin resistant tuf mutations and identified thirteen single amino acid substitutions at sevendifferent sites in EF-Tu. These have been mapped onto the 3D structures of EF-Tu’GTP and EF-Tu.GDP. In the active GTP form of EF-Tu themutations cluster on each side of the interface between domains I and III. We propose that this domain interface is the binding site for kirromycin.

  • 4.
    Abdulkarim, Farhad
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Tuohy, TMF
    Buckingham, RH
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Missense substitutions lethal to essential functions of EF-Tu1991In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 73, no 12, p. 1457-1464Article in journal (Refereed)
    Abstract [en]

    We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.

  • 5.
    Andersson, Dan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkman, Johanna
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Antibiotikaresistens: är den reversibel?1998In: Smittskydd: Smittskyddsinstitutets tidskrift, ISSN 1401-0690, Vol. 4, no 1, p. 3-5Article, book review (Other academic)
  • 6.
    Andersson, D.I
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Björkman, Johanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Antibiotikaresistens här för att stanna?1998In: Läkartidningen, ISSN 0023-7205, E-ISSN 1652-7518, Vol. 95, no 37, p. 3940-3944Article, review/survey (Other academic)
  • 7.
    Andersson, DI
    et al.
    Uppsala University.
    Hughes, Diarmaid
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Muller's Ratchet Impairs Fitness of Bacteria1995Other (Other academic)
  • 8.
    Bilgin, Neş'e
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Kirsebom, Leif A
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Ehrenberg, Måns
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Kurland, Charles G
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Mutations in ribosomal proteins L7/L12 perturb EF-G and EF-Tu functons1988In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 70, no 5, p. 611-618Article in journal (Refereed)
    Abstract [en]

    In vitro cycling rates of E. coli ribosomes and of elongation factors EF-Tu and EF-G have been obtained and these are compatible with translation rates in vivo. We show that the rate of translocation is faster than 50 s-1 and therefore that the EF-G function is not a rate limiting step in protein synthesis. The in vivo phenotype of some L7/L12 mutants could be accounted for by perturbed EF-Tu as well as EF-G functions. The S12 mutants that we studied were, in contrast, only perturbed in their EF-Tu function, while their EF-G interaction was not impaired in relation to wild type ribosomes.

  • 9.
    Bjorkman, J
    et al.
    Uppsala University.
    Hughes, Diarmaid
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Andersson, DI
    Uppsala University.
    Virulence of antibiotic-resistant Salmonella typhimurium1998In: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, ISSN 0027-8424, Vol. 95, no 7, p. 3949-3953Article in journal (Other academic)
    Abstract [en]

    We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice, Of seven resistant mutants examined, sis were avirulent and one was similar to the wild type In competition experiments in mi

  • 10.
    Chaudhuri, Barnali Neel
    Uppsala University, Department of Molecular Biology.
    Structural aspects of binding and transport proteins: By Barnali Neel Chaudhuri1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Binding and transport proteins are essential for the transport of nutrients, vitamins, hormones, pheromones, etc. to their target destinations. Lipid-binding proteins typically contain an internal cavity where the hydrophobic/amphipathic ligands bind. Transport proteins with binding clefts for water-soluble sugars have different architectures. Access to the cavity or the cleft sometimes requires conformational changes in the protein. This thesis presents the structures of three classes of bindingproteins.

    The cellular retinoid binding proteins sequester the amphipathic retinoids (vitamin A analogues) within the cytoplasm. The anti-carcinogenic properties of some synthetic retinoids make them interesting as potential drugs. The structures of cellular retinoic acid binding proteins were determined in complex with three different retinoids. In addition, the structure of a ligand-free mutant of cellular retinol binding protein was determined in order to study ligand entry into the cavity.

    The structure of α2U-globulin, an extra-cellular lipid-binding protein, was obtained in complex with a toxin, d-limonene 1,2-epoxide. The nature of the hydrophobic ligand-binding cavity is described in the context of a male rat specific nephropathy induced by this toxin.

    Allose binding protein from Escherichia coli belongs to the family of periplasmic binding proteins. Structures of this protein in the closed, allose-bound and the open, ligand-free form were determined to study the binding cleft and hinge bending. The protein consists of two Rossmann fold domains joined by a flexible, three-stranded hinge region. The binding cleft is formed at the domain interface in the closed form. Integral water molecules play an important structural role in the hinge.

  • 11.
    Ehrenberg, Måns
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Bilgin, Neş’e
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Dincbas, Vildan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Karimi, Reza
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Abdulkarim, Farhad
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    tRNA-ribosome interactions1995In: Biochemistry and Cell Biology, ISSN 0829-8211, E-ISSN 1208-6002, Vol. 73, no 11-12, p. 1049-1054Article, review/survey (Refereed)
    Abstract [en]

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

  • 12.
    Hansson, Tomas
    Uppsala University, Department of Molecular Biology.
    Ligand binding and enzyme catalysis studied by molecular dynamics simulations1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Molecular dynamics simulations and free energy calculations can be applied to biomolecular systems to predict ligand binding affinities. Combined with hybrid quantum-classical potential energy surfaces, such computations can also be used to probe enzymatic catalysis mechanisms.

    For structure based drug design, an accurate method is needed for binding free energy prediction. A linear interaction energy (LIE) methods is presented, which calculates binding affinities from time averages of interaction potential energies between ligand and surroundings in simulations of the complex of ligand and receptor and of the ligand free in solution. The original version of the method assumes electrostatic linear response of the surrounding medium. The method is applied to inhibitors of HIV-1 proteinase, giving reasonable predictions of inhibition constants.

    Electrostatic linear response of the solvent to the solute electric field is valid for ionic species in aqueous solution, but specific deviations are found for electroneutral dipolar compounds. Statistical investigations using data from 18 complexes with four different proteins show that, in contrast to several other possible extensions to the method, these specific nonlinearities can be utilized to give a real improvement to the predictive power of the LIE method.

    Protein tyrosine phosphatases (PTPases) hydrolyze phosphate esters by formation of acovalent phosphoenzyme intermediate with a cysteine residue. This is followed by hydrolysis of the intermediate. Reaction potential surfaces of different possible steps in intermediate formation in the low molecular weight PTPase are modelled by the empirical valence bond (EVB) method. Molecular dynamics simulations and free energy perturbation calculations then yield reaction free energy profiles for these reactions steps.

    The activation of the cysteine 12 nucleophile is the object of a preliminary study. More extensive EVB investigations and substrate binding calculations yield a reaction free energy profile consistent with experimental findings for the formation of the phosphoenzyme intermediate. The calculations clearly indicate that the reaction complex is protonated and carries a total charge of (-2), and that the leaving group becomes protonated in concert with its expulsion.

  • 13.
    Henriksson, Hongbin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Munoz, Irene
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Isaksson, Roland
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Cellobiohydrolase 58 (P.c. Cel 7D) is complementary to the homologous CBHI (T.r. Cel 7A) in enantioseparations2000In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 898, no 1, p. 63-74Article in journal (Refereed)
    Abstract [en]

    Cellobiohydrolase 58 (EC 3.2.1.91, pc. Cel 7D) from Phanerochaete chrysosporium was immobilized on silica and the resulting material, CBH 58-silica, was then used as a chiral stationary phase (CSP) in liquid chromatographic separations of enantiomers. The enantioselectivities obtained on CBH 58-silica were compared with those on CBH I-silica (a phase based on a corresponding cellulase from Trichoderma reesei). CBH 58-silica displayed higher selectivity than CBH I-silica for the more hydrophilic compounds, such as atenolol and metoprolol, although great similarities in chiral separation of beta -adrenergic antagonists were found between the two phases. None of the acidic compounds tested could be resolved on the CBH 58 phase. Moreover, the solutes were retained more on the CBH 58 phase in general, indicating an improved application potential in bioanalysis. Addition of cellobiose or lactose, both of which are inhibitors of cellulases, To the mobile phase impaired the enantioselectivity, indicating an overlap of the enantioselective and catalytic sites. The chiral analytes also functioned as competitive inhibitors and their inhibition constants were determined.

  • 14.
    Hinas, Andrea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Söderbom, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Treasure hunt in an amoeba: non-coding RNAs in Dictyostelium discoideum.2007In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 51, no 3, p. 141-159Article in journal (Refereed)
    Abstract [en]

    The traditional view of RNA being merely an intermediate in the transfer of genetic information, as mRNA, spliceosomal RNA, tRNA, and rRNA, has become outdated. The recent discovery of numerous regulatory RNAs with a plethora of functions in biological processes has truly revolutionized our understanding of gene regulation. Tiny RNAs such as microRNAs and small interfering RNAs play vital roles at different levels of gene control. Small nucleolar RNAs are much more abundant than previously recognized, and new functions beyond processing and modification of rRNA have recently emerged. Longer non-coding RNAs (ncRNAs) can also have important regulatory roles in the cell, e.g., antisense RNAs that control their target mRNAs. The majority of these important findings arose from analyses in various model organisms. In this review, we focus on ncRNAs in the social amoeba Dictyostelium discoideum. This important genetically tractable model organism has recently received renewed attention in terms of discovery, regulation and functional studies of ncRNAs. Old and recent findings are discussed and put in context of what we today know about ncRNAs in other organisms.

  • 15.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Both genes for EF-Tu in Salmonella typhimurium are individually dispensible for growth1990In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 215, no 1, p. 41-51Article in journal (Refereed)
    Abstract [en]

    Each of the two genes encoding EF-Tu in Salmonella typhimurium has been inactivated using a mini-Mu MudJ insertion. Eleven independently isolated insertions are described, six in tufA and five in tufB. Transduction analysis shows that the inserted MudJ is 100% linked to the appropriate tuf gene. A mutant strain with electrophoretically distinguishable EF-TuA and EF-TuB was used to show, on two-dimensional gels, that the MudJ insertions result in the loss of the appropriate EF-Tu protein. Southern blotting, using cloned Escherichia coli tuf sequences as probes, shows that each MudJ insertion results in the physical breakage of the appropriate tuf gene. The degree of growth-rate impairment associated with each tuf inactivation is independent of which tuf gene is inactivated. The viability of S. typhimurium strains with either tuf gene inactive contrasts strongly with data suggesting that in the closely related bacterium E. coli, an active tufA gene is essential for growth. Finally the strains described here facilitate the analysis of phenotypes associated with individual mutant or wild-type Tus both in vivo and in vitro.

  • 16.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Error-prone EF-Tu reduces in vivo enzyme activity and cellular growth rate1991In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 5, no 3, p. 623-630Article in journal (Refereed)
    Abstract [en]

    Mutations in Salmonella typhimurium encoding error-prone EF-Tu (tufA8, tufB103) enhance translational error levels and also cause a reduced growth rate. The relative changes in error level and growth rate are inversely related and dependent on the status of the two tuf genes. Possible causes of the reduced growth rate were investigated. Several important parameters with the potential to alter growth rate (the EF-Tu-ribosome interaction, the in vivo elongation rate and the processivity of translation), are all relatively unaffected by the tuf mutations. The small reduction in processivity observed in some strains is not quantitatively related to the growth rate reduction. Instead, the error-enhancing mutations are associated with a large reduction in the specific activity of a test protein, β-galactosidase, suggesting by inference that the reduced growth rate is a consequence of the synthesis of error-containing proteins.

  • 17.
    Hughes, Diarmaid
    et al.
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Andersson, DI
    Uppsala University.
    Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates1997In: JOURNAL OF BACTERIOLOGY, ISSN 0021-9193, Vol. 179, no 21, p. 6688-6691Article in journal (Other academic)
    Abstract [en]

    Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells, Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy wer

  • 18.
    Hughes, Diarmaid
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Buckingham, RH
    The nucleotide sequence of rpsL and its flanking regions in Salmonella typhimurium1991In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 104, no 1, p. 123-124Article in journal (Refereed)
    Abstract [en]

    The ribosomal protein (r-protein)-encoding gene, rpsL, and regions flanking it, from Salmonella typhimurium, have been sequenced directly from polymerase chain reaction-amplified chromosomal DNA. The deduced amino acid sequence is identical to that of the Escherichia coli rpsL encoded r-protein. At the nucleotide level, the similarity is 98%, suggesting a strong pressure for the conservation of this important protein. More surprisingly, the noncoding sequences surrounding the gene are also conserved at the 98% level, suggesting that they too are functionally important.

  • 19.
    Johanson, U
    et al.
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    AEvarsson, A
    Liljas, A
    Hughes, Diarmaid
    Uppsala University. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    The dynamic structure of EF-G studied by fusidic acid resistance and internal revertants1996In: JOURNAL OF MOLECULAR BIOLOGY, ISSN 0022-2836, Vol. 258, no 3, p. 420-432Article in journal (Other academic)
    Abstract [en]

    We have previously identified 20 different fusidic acid-resistant alleles of fusA, encoding mutant forms of the ribosomal translocase EF-G. One of these, P413L, is used here as the starting point in selections for internal revertants, identifying 20 diffe

  • 20.
    Johanson, Urban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    A new mutation in 16S rRNA of Escherichia coli conferring spectinomycin resistance1995In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 23, no 3, p. 464-466Article in journal (Refereed)
    Abstract [en]

    We report a novel mutation, Cl 066U in 16S rRNA whichwas selected for resistance to spectinomycin, anantibiotic which inhibits ribosomal translocation. Theminimal inhibitory concentration (MIC) of spectinomycindetermined for this mutant (15 pg/ml) is greaterthan with the wild-type plasmid (5 ig/ml) but lower thanwith the well known C1192U mutation (>80 pg/ml). TheCl 066U mutation also increases the cells sensitivity tofusidic acid, another antibiotic which inhibits translationat the translocation stage, whereas C1192U isunchanged relative to the wild type. We discuss whythe acquisition of resistance to one of these drugs isoften associated with hypersensitivity to the other.

  • 21.
    Johanson, Urban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Comparison of the complete sequence of the str operon in Salmonella typhimurium and Escherichia coli1992In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 120, no 1, p. 93-98Article in journal (Refereed)
    Abstract [en]

    The nucleotide (nt) sequences of the str operon in Escherichia coli K-12 and Salmonella typhimurium LT2 were completed and compared at the nt and amino acid (aa) level. The order of conservation at the nt and aa level is rpsL > tufA > rpsG > fusA. A striking difference is that the rpsG-encoded ribosomal protein, S7, in E. coli K-12 is 23 aa longer than in S. typhimurium. The very low (0.18) codon adaptation index of this part of the E. coli K-12-encoding gene and the unusual stop codon (UGA) suggest that this is a relatively recent extension. A trend towards a higher G+C content in fusA (gene encoding elongation factor (EF)-G) and tufA (gene encoding EF-Tu) in S. typhimurium is noted. In fusA, nt substitutions at all three positions in a codon occur at a much higher frequency than expected from the number of nt substitutions in the gene, assuming they are random and independent events. An analysis of substitutions in this and other genes suggests that the triple substitutions in fusA, and some other genes, are the result of the sequential accumulation of individual mutations, probably driven by selection pressure for particular codons or aa.

  • 22.
    Johanson, Urban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Fusidic acid-resistant mutants define three regions in elongation factor G of Salmonella typhimurium1994In: Gene, ISSN 0378-1119, E-ISSN 1879-0038, Vol. 143, no 1, p. 55-59Article in journal (Refereed)
    Abstract [en]

    We have sequenced fusA, the gene coding for elongation factor G (EF-G) in 18 different mutants of Salmonella typhimurium selected as fusidic acid resistant (Fu(R). In addition, we have sequenced two previously described Fu(R) mutants from Escherichia coli. In all cases, the resistance is due to a mutation in one of three separate regions in fusA. The three clusters of mutant sites superimpose on regions that are well conserved, suggesting that they are of a more general functional importance. To further classify the mutants, we have measured the minimal inhibitory concentration (MIC) for Fu and for two other antibiotics which interfere with translocation on the ribosome. kanamycin (Km) and spectinomycin (Sp). The levels of resistance to Fu for each of the mutants are significantly higher than in the wild type (wt), and vary by about one order of magnitude between the highest and the lowest. Most of the mutants are also more resistant to Km than the wt, although the level of resistance is low and the variation small. In contrast, about half of the mutants are more sensitive to Sp than the wt, with only one being more resistant. Only three of the twenty mutants behave like the wt with respect to the non-selected phenotypes, Km(R) and Sp(R).

  • 23.
    Karimi, Reza
    Uppsala University, Department of Molecular Biology.
    Determinants of peptidyl-tRNA drop-off from E. coli Ribosomes: Effects of antibiotics, mutations and translation factors1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    During protein synthesis, some peptidyl-tRNA molecules dissociate from the ribosomes and become hydrolysed by peptidyl-tRNA hydrolase (Pth). Two different in vitro systems, poly(U) and natural mRNA programmed ribosomes, have been used in order to determine dissociation (drop-off) rates of deacylated- and peptidyl-tRNAs from different sites on ribosomes.

    A direct measurement of the rates of dissociation of dipeptidyl-tRNAs from the A- and P-site of poly(U) programmed wild-type (wt), hyper-accurate (SmD and SmP, altered in ribosomal protein S12) and error-prone (ram, altered in ribosomal protein S4 or S5) ribosomes has been performed. Dipeptidyl-tRNA dissociates faster from the A-site of wt than from ram ribosomes and it dissociates faster from SmD and SmP than from wt ribosomes. The dissociation of dipeptidyl-tRNA from the P-site has an opposite behaviour compared to the A-site. Dipeptidyl-tRNA dissociates faster from the P-site of ram than from wt ribosomes and faster from wt than SmD and SmP ribosomes. Aminoglycoside antibiotics [streptomycin (Sm)and neomycin (Nm)] reduce the dissociation rate of dipeptidyl-tRNA from the A-site and increase it from the P-site for all these ribosomal variants. Remarkably, when the binding of dipeptidyl-tRNA to the A-site is weak, the affinity for the P-site is strong, and vice versa. In the absence of Sm non-cognate dipeptidyl-tRNA dissociates 150 times faster from the A-site of ram ribosomes than cognate. In the presence of Sm the rate difference is only eight fold. This suggests that Sm may distort the 16S rRNA conformation and, consequently, the codon-anticodon structure.

    Using a new in vitro system with heteropolymeric mRNAs has revealed that i) Initiation factors IF1 and IF2 synergistically remove short peptidyl-tRNAs from the P-site of ribosomes in a way that depends both on the length and sequence of the nascent peptide. ii) Ribosome recycling factor (RFF), elongation factor G (EF-G) and release factor 3 (RF3) stimulate dissociation of short peptidyl-tRNAs from the P-site in almost the same manner as IF1/IF2. iii) RRF, EF-G, GTP and initiation factor 3 (IF3), after termination, split the 70S ribosomes and remove deacylated tRNA from the P-site of ribosomes in a mechanism that is strictly dependent on GTP hydrolysis. In vivo experiments, showing that overexpression of RRF or simultaneous overexpression of IF1/IF2 impairs growth rate in thermosensitive pth mutant strains, are consistent with the in vitro results.

  • 24.
    Kurland, CG
    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, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Ehrenberg, M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Limitations of translational accuracy1996In: Escherichia coli and Salmonella: Cellular and Molecular Biology, ASM Press, Washington DC, USA , 1996, Vol. 1, p. 979-1004Chapter in book (Other academic)
  • 25.
    Rydén Aulin, Monica
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Overproduction of release factor reduces spontaneous frameshifting and frameshift suppression by mutant elongation factor Tu1990In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 172, no 12, p. 6721-6726Article in journal (Refereed)
    Abstract [en]

    Mutant forms of elongation factor Tu encoded by tufA8 and tufB103 in Salmonella typhimurium cause suppression of some but not all frameshift mutations. All of the suppressed mutations in S. typhimurium have frameshift windows ending in the termination codon UGA. Because both tufA8 and tufB103 are moderately efficient UGA suppressors, we asked whether the efficiency of frameshifting is influenced by the level of misreading at UGA. We introduced plasmids synthesizing either one of the release factors into strains in which the tuf mutations suppress a test frameshift mutation. We found tht overproduction of release factor 2 (which catalyzes release at UGA and UAA) reduced frameshifting promoted by the tuf mutations at all sites tested. However, at one of these sites, trpE91, overproduction of release factor 1 also reduced suppression. The spontaneous level of frameshift 'leakiness' at three sites in trpE, each terminating in UGA, was reduced in strains carrying the release factor 2 plasmid. We conclude that both spontaneous and suppressor-enhanced reading-frame shifts are influenced by the activity of peptide chain release factors. However, the data suggest that the effect of release factor on frameshifting does not necessarily depend on the presence of the normal triplet termination signal.

  • 26.
    Sigrell, Jill A.
    Uppsala University, Department of Molecular Biology.
    A structural study of ribokinase from Escherichia coli1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ribokinase phosphorylates the abundant sugar ribose, using ATP in the presence of magnesium. The phosphorylation is the first reaction in ribose metabolism and the product, ribose-5-phosphate, can enter the pentose phosphate pathway and glycolysis. It can also be used in the synthesis of histidine and purines. Ribokinase belongs to the ribokinase family which includes various carbohydrate kinases from both prokaryotes and eukaryotes. No member's three-dimensional structure has previously been determined.

    In this thesis the threedimensional structure of ribokinase from Escherichia coli, determined by X-ray crystallography, is presented. The enzyme is a dimer and each subunit consists of a large a/b domain and a small b-sheet domain. The two b-sheet domains interact and form a b-clasp at the dimer interface. The two active sites are found in a cleft between the b-clasp and the a/b domains.

    Further structural studies on the enzyme in the apo form and in binary and ternary complexes show that the enzyme has both open and closed states. Ribose induces the conformational change into the closed form. The small b-sheet domain covers the ribose-binding site as a lid and produces a good micro environment for the phosphorylation reaction.

    The ordered reaction is thought to involve a catalytic acid (Asp255) and go through an in-line mechanism where the ribose O5* is the nucleophile attacking the g-phosphate of the ATP. The penta-covalent transition state can be stabilised by a magnesium ion, an anion hole and a lysine side chain. Preliminary kinetic studies have determined the KM for ribose to be 0.2 mM. These results may be applicable to other members of the family.

  • 27.
    Ståhlberg, Jerry
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Henriksson, Hongbin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Divne, Christina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Isaksson, Roland
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Chemistry.
    Pettersson, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Johansson, Gunnar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Alwyn, Jones T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 angstrom resolution.2001In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 305, no 1, p. 79-93Article in journal (Refereed)
    Abstract [en]

    Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta -blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers.

    In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta -blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1.9 Angstrom resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/ + 1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O-epsilon1 and to Gln175 N-epsilon2, whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q.

    Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 muM and 44 muM, respectively, corresponding to binding free energies of 20 kJ/ mol and 24 kJ/mol, respectively. The K-i value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed.

    By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non-selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP

  • 28.
    Tubulekas, I
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome1993In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 175, no 1, p. 240-250Article in journal (Refereed)
    Abstract [en]

    Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.

  • 29.
    Tubulekas, Ioannis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Buckingham, Richard H.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Mutant ribosomes can generate dominant kirromycin resistance1991In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 173, no 12, p. 3635-3643Article in journal (Refereed)
    Abstract [en]

    Mutations in the two genes for EF-Tu in Salmonella typhimuriumand Escherichia coli, tufA and tufB, can confer resistance tothe antibiotic kirromycin. Kirromycin resistance is a recessivephenotype expressed when both tuf genes are mutant. We describea new kirromycin-resistant phenotype dominant to the effectof wild-type EF-Tu. Strains carrying a single kirromycin-resistanttuf mutation and an error-restrictive, streptomycin-resistantrpsL mutation are resistant to high levels of kirromycin, evenwhen the other tuf gene is wild type. This phenotype is dependenton error-restrictive mutations and is not expressed with nonrestrictivestreptomycin-resistant mutations. Kirromycin resistance is alsoexpressed at a low level in the absence of any mutant EF-Tu.These novel phenotypes exist as a result of differences in theinteractions of mutant and wild-type EF-Tu with the mutant ribosomes.The restrictive ribosomes have a relatively poor interactionwith wild-type EF-Tu and are thus more easily saturated withmutant kirromycin-resistant EF-Tu. In addition, the mutant ribosomesare inherently kirromycin resistant and support a significantlyfaster EF-Tu cycle time in the presence of the antibiotic thando wild-type ribosomes. A second phenotype associated with combinationsof rpsL and error-prone tuf mutations is a reduction in thelevel of resistance to streptomycin.

  • 30.
    Tubulekas, Ioannis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Growth and translation elongation rate are sensitive to the concentration of EF-Tu1993In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 8, no 4, p. 761-770Article in journal (Refereed)
    Abstract [en]

    We have used quantitative immunoblotting to estimate the amount of EF-Tu in a variety of S. typhimurium strains with wild-type, mutant, insertionally inactivated or plasmid-borne tuf genes. In the same strains we have measured translation elongation rate, exponential growth rate and the level of nonsense codon readthrough. In the wild-type strain, at moderate to fast growth rates, our data show that EF-Tu makes up 8–9% of total cell protein. Strains with either of the tuf genes insertionally inactivated have 65% of the wild-type EF-Tu level, irrespective of which tuf gene remains active, or whether that gene is wild-type or a kirromycin-resistant mutant. Strains with only one active tuf gene have reduced growth and translation elongation rates. From the magnitude of the reduction in elongation rate relative to the level of EF-Tu we calculate that in glucose minimal medium the in vivo saturation level of wild-type ribosomes by ternary complexes is only 63%. Strains with a ribosome mutation causing a poor interaction with ternary complex are non-viable on minimal medium when the level of EF-Tu is reduced.

  • 31.
    Tubulekas, Ioannis
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Suppression of rpsL phenotypes by tuf mutations reveals a unique relationship between translation elongation and growth rate1993In: Molecular Microbiology, ISSN 0950-382X, Vol. 7, no 2, p. 275-284Article in journal (Refereed)
    Abstract [en]

    We have found a simple relationship between bacterial growth rate and the translation elongation rate. Thus, for a set of defined ribosomal protein S12 mutations which reduce the efficiency of the ternary complex ribosome interaction (and restrict the frequency of translational errors) there is a linear relationship between growth rate and translation elongation rate. When these mutants are combined with defined EF-Tu mutants (which increase the probability of translational errors) both the elongation rate and growth rate reductions are reversed. The reductions and reversals are described by a unique linear relationship. We interpret this to mean that these two types of mutation exert opposing effects on the same molecular interaction. We suggest that this interaction is in the initial selection of the aminoacyl-tRNA on the ribosome. The slope of the relationship between translation elongation rate and growth rate, defined in per cent of the wild-type rates, is close to 1. Interestingly, the reversal of the elongation and growth phenotypes is Incomplete, suggesting that the ribosomal mutants have an additional defect which is not compensated for by the ternary complex interaction. Our results show that the efficiency of the ternary complex ribosome interaction limits the translation elongation rate, which in turn correlates with changes in exponential growth rate.

  • 32. Tuohy, TMF
    et al.
    Thompson, S
    Gesteland, R
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Atkins, JF
    The role of EF-Tu and other translation components in determining translocation step size1990In: Biochimica et Biophysica Acta, Gene Structure and Expression, ISSN 0167-4781, E-ISSN 1879-2634, Vol. 1050, no 1-3, p. 274-278Article in journal (Refereed)
    Abstract [en]

    The two EF-Tu encoding genes, tufA and tufB, of Salmonella typhimurium have been sequenced. Nearly all the differences from their Escherichia coli counterparts are third position changes which do not alter the encoded amino acids. Unexpectedly, most of the changes in one Salmonella tuf gene are paralleled by changes in the other tuf gene perhaps due to gene repair despite the distance separating the genes. Three mutants which cause mis-framing, have their substitutions at codon 375. Explanations for mutants which cause mis-framing are considered and the mechanism of normal reading frame maintenance discussed.

  • 33.
    Zomorodipour, Alireza
    Uppsala University, Department of Molecular Biology.
    Mapping the Rickettsia prowazekii genome1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Rickettsia prowazekii is a member of alpha proteobacteria and the etiologic agent of epidemictyphus. Besides its pathogenicity as an obligate intracellular parasite it has been considered as amodel organism in a variety of studies.

    We have established a collection of overlapping clones, covering nearly the complete R.prowazekii genome. The clones were isolated from three genomic libraries in cosmid, lambdaphage and lambda-Zap vectors and arranged along the entire genome, using a bottom-upmethod. The ordered genomic libraries have served an important function in the sequencing ofthe entire genome and will be useful for further functional studies of the R. prowazekii genes.

    The sequencing and analysis of the entire R. prowazekii genome have provided evidencefor an exclusive obligate intracellular parasite with massive genomic reduction and a generalgenomic rearrangement in addition to an unusually high fraction (24%) of non-coding regions.There is evidence to suggest that the non-coding regions may be remnants of genes which areeliminated from the genome in an on-going process of genomic reduction. The genomicrearrangements are likely to have occurred through intra-chromosomal recombination events,mediated by repeated sequences in the ancestral genome which might have been deletedsubsequently. The rearrangement of the genome was evidenced in our earlier work whichdetected that the single copy of the 16S rRNA gene is located distantly from the linked 23S-5SrRNA genes in the R. prowazekii genome.

    Phylogenetic analysis based on a large number of R. prowazekii genes indicates that R.prowazekii is more closely related to mitochondria than any other microbial genome. Theobtained results are in accordance with the previously obtained data which was based onphylogentic analysis of rRNA genes. The partial similarities in gene composition between R.prowazekii and mitochondrial genomes is also additional evidence for this relationship.

    A comparative analysis between the complete genomes of the R. prowazekii and theChlamydia trachomatis, a distantly related obligate intracellular parasite, uncovered a total of325 genes with significant homology between the two genomes. A similar pattern of reductionin the corresponding functional categories is observed between these two genomes. Duringadaptation to their eukaryotic nutrient-rich environment, the two parasitic bacteria haveundergone a massive genomic loss and became more dependent on a host. In compensation,they have developed systems such as transporters that can exploit host cell provided nutrients.The comparative analysis has provided additional evidence for a convergent evolution duringadaptation to an intracellular life style in two distantly related bacteria.

  • 34.
    Zou, JY
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Jones, TA
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Towards the automatic interpretation of macromolecular electron-density maps: Qualitative and quantitative matching of protein sequence to map1996In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 52, p. 833-841Article in journal (Refereed)
    Abstract [en]

    The matching of the known polypeptide sequence to the electron density is a critical step in solving protein structures by the crystallographic method. Tools have been developed to help in defining the placement of the sequence, both qualitatively and quantitatively. They have been tested with good results on two proteins whose structures were solved by the MIR method.

  • 35.
    Åqvist, Johan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Molecular Biology.
    Mowbray, Sherry L.
    Sugar Recognition by a Glucose/Galactose Receptor: Evaluation of Binding Energetics from Molecular Dynamics Simulations1995In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 270, no 17, p. 9978-9981Article in journal (Refereed)
    Abstract [en]

    A new theoretical method for free energy calculations is used to compute the absolute binding constants for β-D-glucose and methyl-β-D-galactoside to the periplasmic glucose/galactose receptor from Salmonella typhimurium. The computer simulation results agree well with available experimental data and make it possible to assess the sources of both the high affinity as well as the specificity for glucose. It was found that the major contribution to the binding energy comes from electrostatic interactions and particularly hydrogen bonds of the charge-dipole type. We also predict the structure of the complex with methyl-galactoside as this has not yet been experimentally determined.

1 - 35 of 35
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