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Thomas L. James - One of the best experts on this subject based on the ideXlab platform.
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High-resolution NMR structure of an AT-rich DNA sequence
Journal of Biomolecular NMR, 2002Co-Authors: Nikolai B. Ulyanov, William R. Bauer, Thomas L. JamesAbstract:We have determined, by proton NMR and complete relaxation matrix methods, the high-resolution structure of a DNA oligonucleotide in solution with nine contiguous AT base pairs. The stretch of AT pairs, TAATTATAA⋅TTATAATTA, is imbedded in a 27-nucleotide stem-and-loop construct, which is stabilized by terminal GC base pairs and an extraordinarily stable DNA loop GAA (Hirao et al., 1994, Nucleic Acids Res. 22 , 576–582). The AT-rich sequence has three repeated TAA⋅TTA motifs, one in the reverse orientation. Comparison of the local conformations of the three motifs shows that the sequence context has a minor effect here: atomic RMSD between the three TAA⋅TTA fragments is 0.4–0.5 Å, while each fragment is defined within the RMSD of 0.3–0.4 Å. The AT-rich stem also contains a consensus sequence for the Pribnow Box, TATAAT. The TpA, ApT, and TpT⋅ApA steps have characteristic local conformations, a combination of which determines a unique sequence-dependent pattern of minor groove width variation. All three TpA steps are locally bent in the direction compressing the major groove of DNA. These bends, however, compensate each other, because of their relative position in the sequence, so that the overall helical axis is essentially straight.
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probability assessment of conformational ensembles sugar repuckering in a dna duplex in solution
Biophysical Journal, 1995Co-Authors: Nikolai B. Ulyanov, Uli Schmitz, Anil Kumar, Thomas L. JamesAbstract:Conformational flexibility of molecules in solution implies that different conformers contribute to the NMR signal. This may lead to internal inconsistencies in the 2D NOE-derived interproton distance restraints and to conflict with scalar coupling-based torsion angle restraints. Such inconsistencies have been revealed and analyzed for the DNA octamer GTATAATG.CATATTAC, containing the Pribnow Box consensus sequence. A number of subsets of distance restraints were constructed and used in the restrained Monte Carlo refinement of different double-helical conformers. The probabilities of conformers were then calculated by a quadratic programming algorithm, minimizing a relaxation rate-base residual index. The calculated distribution of conformers agrees with the experimental NOE data as an ensemble better than any single structure. A comparison with the results of this procedure, which we term PARSE (Probability Assessment via Relaxation rates of a Structural Ensemble), to an alternative method to generate solution ensembles showed, however, that the detailed multi-conformational description of solution DNA structure remains ambiguous at this stage. Nevertheless, some ensemble properties can be deduced with confidence, the most prominent being a distribution of sugar puckers with minor populations in the N-region and major populations in the S-region. Importantly, such a distribution is in accord with the analysis of independent experimental data--deoxyribose proton-proton scalar coupling constants.
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solution structure of a dna octamer containing the Pribnow Box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two dimensional nuclear magnetic resonance spectral fitting
Journal of Molecular Biology, 1992Co-Authors: Uli Schmitz, Ingmar Sethson, William Egan, Thomas L. JamesAbstract:Abstract The DNA octamer [d(GTATAATG] · [d(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5′ and H5″ protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY). and T 1ϱ measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities. MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA. B-DNA , and wrinkled D-DNA , another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini. A detailed analysis of the conformation of the final structure and comparison with the structure of a related sequence. [d(GTATATAC)] 2 reported earlier, is described.
L K Siu - One of the best experts on this subject based on the ideXlab platform.
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[[alternative]]High-Level Expression of AmpC ß-Lactamase Due to Insertion of Nucleotides between -10 and -35 Promoter Sequences in Escherichia coli Clinical Isolates: Cases Not Responsive to Extended-Spectrum-Cephalosporin Treatment
'American Society for Microbiology', 2008Co-Authors: L K SiuAbstract:[[abstract]]Two Escherichia coli isolates were recovered from the blood of two cancer patients and were demonstrated to produce high levels of the AmpC beta-lactamase with isoelectric points of >9.0. The hypertranscription of ampC RNA was observed by Northern blot hybridization in both isolates. One isolate (isolate EC44) had a point mutation (G-->A at position -28) and insertion of thymidine between positions -20 and -19 of the ampC promoter gene (GenBank accession no. AE000487). The single nucleotide insertion of T between positions -19 and -20 created an optimal distance (17 bp) in the Pribnow Box for ampC hyperproduction. The other isolate (isolate EC38) had two point mutations (G-->A at position -28 and C-->T at position +58) and a 2-base (GT) insertion between positions -14 and -15. Although the insertion of GT between positions -14 and -15 may create a new promoter next to the original promoter, cloning of the ampC region with truncated nucleotides of the original -35 region of EC38 failed to verify the hypothesis that a new promoter would be created by such a nucleotide insertion. Instead, multiple start sites for ampC transcription at -1, +1, +2, and +3 were observed in an S1 nuclease protection assay. These results suggest that the RNA polymerase is flexible in the selection of a start site in ampC hypertranscription. In conclusion, nucleotide insertions between the -35 and -10 ampC promoter sequences was the mechanism for the hyperproduction of AmpC P-lactamase and resistance to oxyimino-cephalosporins. The failure of the two patients to respond to treatment with oxyimino-cephalosporins highlights the important role of such a resistance mechanism in the clinical setting
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transferable hyperproduction of tem 1 beta lactamase in shigella flexneri due to a point mutation in the Pribnow Box
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: L K Siu, Kwokyung Yuen, Samson S Y Wong, P Y ChauAbstract:Hyperproductionof b-lactamaseswasfoundtobeanimportant cause of resistance to b-lactam‐b-lactamase inhibitors among clinical isolates of the family Enterobacteriaceae. For example, hyperproduction of the plasmid-encoded b-lactamase TEM-1 was reported to be the predominant mechanism of resistance to amoxicillin-clavulanate in about 60% ofEscherichia coli strains (13, 19). Three mechanisms may lead to hyperproduction. First, an increase in the gene dosage as a result of repetitions of b-lactamase genes; e.g., repetition of thewild-typeallelesofthegeneencodingAmpC(17),hasbeen known to confer a high level of resistance to amoxicillin-clavulanate. Second, multiple copies of the blaTEM gene due to the presence of multiple copies of plasmid per cell have been reported to cause hyperproduction of b-lactamase (21). Third, hyperproduction of enzyme due to various genetic events affecting the promoter region has been widely reported in various bacteria (2, 3, 10), although this mechanism was much less studied for b-lactamases. We have isolated in Hong Kong twoShigellaflexneristrains resistant to ampicillin-sulbactam due to the hyperproduction of TEM-1 b-lactamase, the gene of which was encoded on a large 120-kb plasmid and was transferable toE. coliby conjugation. The purpose of the present study was to find the molecular basis of TEM-1 hyperproduction by isolating and analyzingtheplasmidDNAandsequencingthe b-lactamasegenes from the two strains. The results thus obtained were compared with those for a TEM-1-producing, ampicillin-sulbactam-susceptible S. flexneri strain in which the blaTEM gene was also found to be encoded on a 120-kb conjugatively transferable plasmid. The strains used in this study and their characteristics are listedinTable1.TheMICsofampicillin,ampicillin-sulbactam, amoxicillin, and amoxicillin-clavulanate were determined by the agar dilution method (15). The ampicillin-sulbactam and amoxicillin-clavulanate combinations were tested at a fixed ratio of 2:1. Antibiotic powders were kindly provided by Pfizer Corporation, Hong Kong (ampicillin and sulbactam), and SmithKline Beecham, Hong Kong (amoxicillin and clavulanate). Susceptibility testing of sulfamethoxazole-trimethoprim, chloramphenicol, tetracycline, and rifampin were performed by the Kirby-Bauer disk diffusion method (16). A rifampin-resistantE. colistrain, strain JP-995 (kindly supplied
Benedicte Fournier - One of the best experts on this subject based on the ideXlab platform.
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strength and regulation of the different promoters for chromosomal β lactamases of klebsiella oxytoca
Antimicrobial Agents and Chemotherapy, 1999Co-Authors: Benedicte Fournier, Annie Gravel, David C Hooper, Paul H RoyAbstract:The two groups of chromosomal beta-lactamases from Klebsiella oxytoca (OXY-1 and OXY-2) can be overproduced 73- to 223-fold, due to point mutations in the consensus sequences of their promoters. The different versions of promoters from blaOXY-1 and blaOXY-2 were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene of pKK232-8, and their relative strengths were determined in Escherichia coli and in K. oxytoca. The three different mutations in the OXY beta-lactamase promoters resulted in a 4- to 31-fold increase in CAT activity compared to that of the wild-type promoter. The G-->T transversion in the first base of the -10 consensus sequence caused a greater increase in the promoter strength of the wild-type promoter than the two other principal mutations (a G-to-A transition of the fifth base of the -10 consensus sequence and a T-to-A transversion of the fourth base of the -35 sequence). The strength of the promoter carrying a double mutation (transition in the Pribnow Box and the transversion in the -35 hexamer) was increased 15- to 61-fold in comparison to that of the wild-type promoter. A change from 17 to 16 bp between the -35 and -10 consensus sequences resulted in a ninefold decrease of the promoter strength. The expression of the blaOXY promoter in E. coli differs from that in K. oxytoca, particularly for promoters carrying strong mutations. Furthermore, the blaOXY promoter appears not to be controlled by DNA supercoiling or an upstream curved DNA, but it is dependent on the gene copy number.
K Hiratsuka - One of the best experts on this subject based on the ideXlab platform.
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cloning and sequence analysis of two copies of a 23s rrna gene from helicobacter pylori and association of clarithromycin resistance with 23s rrna mutations
Antimicrobial Agents and Chemotherapy, 1997Co-Authors: Diane E Taylor, Zhongming Ge, Dale Purych, T Lo, K HiratsukaAbstract:In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow Box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.
Seth A Darst - One of the best experts on this subject based on the ideXlab platform.
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Pribnow Box recognition and melting by escherichia coli rna polymerase
1997Co-Authors: Seth A Darst, Jeffrey W Roberts, Arun Malhotra, Michael T Marr, Konstantin Severinov, Elena SeverinovaAbstract:The core RNA polymerases from bacterial and eukaryotic cells, which are homologous in structure and function (Allison et al. 1985; Biggs et al. 1985; Ahearn et al. 1987; Sweetser et al. 1987; Darst et al. 1989, 1991; Schultz et al. 1993; Polyakov et al. 1995), are catalytically active in RNA chain elongation but are incapable of promoter recognition and specific initiation. Promoter-specific transcription initiation requires additional protein factors. In bacteria, specific initiation by RNA polymerase (RNAP) requires a single polypeptide known as a σ factor, which binds to core RNAP to form the holoenzyme (Burgess et al. 1969; Travers and Burgess 1969). One primary σ factor directs the bulk of transcription during exponential growth. Specialized, alternative σ factors direct transcription of specific regulons during unusual physiological or developmental conditions (reviewed in Helmann and Chamberlin 1988; Gross et al. 1992). The primary and most of the alternative σ factors comprise a highly homologous family of proteins (Stragier et al. 1985; Gribskov and Burgess 1986) with four regions of highly conserved amino acid sequence (Fig. 1; reviewed in Lonetto et al. 1992). Based on the results of genetic and biochemical experiments, specific functions have been assigned to some of the conserved regions(summarized in Fig. 1).
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crystal structure of a σ70 subunit fragment from e coli rna polymerase
Cell, 1996Co-Authors: Arun Malhotra, Elena Severinova, Seth A DarstAbstract:Abstract The 2.6 A crystal structure of a fragment of the σ 70 promoter specificity subunit of E. coli RNA polymerase is described. Residues involved in core RNA polymerase binding lie on one face of the structure. On the opposite face, aligned along one helix, are exposed residues that interact with the −10 consensus promoter element (the Pribnow Box), including four aromatic residues involved in promoter melting. The structure suggests one way in which DNA interactions may be inhibited in the absence of RNA polymerase and provides a framework for the interpretation of a large number of genetic and biochemical analyses.