Fine mapping of DNA single-stranded regions using base-specific chemical probes: study of an open complex formed between RNA polymerase and the lac UV5 promoter.


:We have used diethyl pyrocarbonate (DEP), which carbethoxylates adenine bases, and dimethyl sulfate (DMS), which methylates guanine residues and single-stranded cytosines, to probe bases in open complexes between RNA polymerase and the lac UV5 promoter in vitro. We compared the kinetics of reactivity between bases in an open complex and those in a single-stranded 35-mer fragment corresponding to the lower template strand of lac UV5 in the region -25 to +10 relative to the transcription start site. We observed that cytosine and adenine residues in the 35-mer fragment reacted according to a second-order process with DMS and DEP, respectively, at sufficiently low concentrations of the reagents and that the degree of reactivity was base position independent. In an open complex in the absence of substrates, we observed reactivity with DEP in adenines from -12 to +4 as well as +21 on the template strand and methylation by dimethyl sulfate of cytosines -6, -4, -2, and -1. No hyperreactivity was observed on the nontemplate strand. The degree of reactivity of bases between -12 and +4 was position dependent, maximum reactivity being displayed by bases in the middle of the region. The reaction was first order within the range of reagent concentration investigated. It was confirmed that in the presence of ApA and UTP cytosine +5, as well as cytosines -6, -4, -2, and -1, in an open complex became reactive to DMS. With regard to DEP the extent of reactivity of the adenine at position +3 was increased markedly, adenine +4 was brought into the single-stranded region, and the overall reactivity of adenine -10 decreased.(ABSTRACT TRUNCATED AT 250 WORDS)






Buckle M,Buc H




Has Abstract


1989-05-16 00:00:00












  • Structures of enterococcal glycerol kinase in the absence and presence of glycerol: correlation of conformation to substrate binding and a mechanism of activation by phosphorylation.

    abstract::The first structure of a glycerol kinase from a Gram-positive organism, Enterococcus casseliflavus, has been determined to 2.8 A resolution in the presence of glycerol and to 2.5 A resolution in the absence of substrate. The substrate-induced closure of 7 degrees is significantly smaller than that reported for hexokin...


    pub_type: 杂志文章


    authors: Yeh JI,Charrier V,Paulo J,Hou L,Darbon E,Claiborne A,Hol WG,Deutscher J

    更新日期:2004-01-20 00:00:00

  • pH dependence and structural interpretation of the reactions of Coprinus cinereus peroxidase with hydrogen peroxide, ferulic acid, and 2,2'-azinobis.

    abstract::Steady-state and transient-state analysis of Coprinus cinereus peroxidase, CIP (identical to Arthromyces ramosus peroxidase), was used to characterize the kinetics of the three fundamental steps in heme peroxidase catalysis: compound I (cpd I) formation, cpd I reduction, and compound II (cpd II) reduction. The rate co...


    pub_type: 杂志文章


    authors: Abelskov AK,Smith AT,Rasmussen CB,Dunford HB,Welinder KG

    更新日期:1997-08-05 00:00:00

  • Insertion and orientation of a synthetic peptide representing the C-terminus of the A1 domain of Shiga toxin into phospholipid membranes.

    abstract::Shiga toxin is a bacterial protein composed of one A and five B subunits. Its A chain possesses a protease sensitive loop (Cys-242-Cys-261) that is cleaved to produce an enzymatically active A1 domain and an A2 fragment associated with its B subunit pentamer. The proposed mode of action of the toxin is linked to its r...


    pub_type: 杂志文章


    authors: Saleh MT,Ferguson J,Boggs JM,Gariépy J

    更新日期:1996-07-23 00:00:00

  • Maduropeptin: an antitumor chromoprotein with selective protease activity and DNA cleaving properties.

    abstract::Maduropeptin (MDP) is a recently isolated antitumor antibiotic, consisting of an enediyne-containing chromophore embedded in a highly acidic protein. This holoantibiotic damages duplex DNA in vitro, producing a mixture of single- and double-strand breaks at selected sites. The chromophore, isolated as the methanol add...


    pub_type: 杂志文章


    authors: Zein N,Solomon W,Colson KL,Schroeder DR

    更新日期:1995-09-12 00:00:00

  • Use of cytochrome P-450scc to measure cholesterol-lipid interactions.

    abstract::The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability i...


    pub_type: 杂志文章


    authors: Stevens VL,Lambeth JD,Merrill AH Jr

    更新日期:1986-07-29 00:00:00

  • Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain.

    abstract::Conventional isoforms of protein kinase C (PKC) are activated when their two membrane-targeting modules, the C1 and C2 domains, bind the second messengers diacylglycerol (DG) and Ca2+, respectively. This study investigates the mechanism of Ca2+-induced binding of PKC betaII to anionic membranes mediated by the C2 doma...


    pub_type: 杂志文章


    authors: Nalefski EA,Newton AC

    更新日期:2001-11-06 00:00:00

  • Amphipathic polymers: tools to fold integral membrane proteins to their active form.

    abstract::Among the major obstacles to pharmacological and structural studies of integral membrane proteins (MPs) are their natural scarcity and the difficulty in overproducing them in their native form. MPs can be overexpressed in the non-native state as inclusion bodies, but inducing them to achieve their functional three-dim...


    pub_type: 杂志文章


    authors: Pocanschi CL,Dahmane T,Gohon Y,Rappaport F,Apell HJ,Kleinschmidt JH,Popot JL

    更新日期:2006-11-28 00:00:00

  • Mapping the suramin-binding sites of human neutrophil elastase: investigation by fluorescence resonance energy transfer and molecular modeling.

    abstract::Neutrophil elastase (NE), a mediator of inflammation, binds with high affinity numerous anionic molecules including suramin, a polysulfated naphthylurea, which inhibits it with a Ki of 0.2 microM and a 4:1 suramin:NE stoichiometry and thus constitutes a potential therapeutic agent. In an attempt to locate the suramin ...


    pub_type: 杂志文章


    authors: Mély Y,Cadène M,Sylte I,Bieth JG

    更新日期:1997-12-16 00:00:00

  • Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis.

    abstract::Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium t...


    pub_type: 杂志文章


    authors: Green KD,Biswas T,Chang C,Wu R,Chen W,Janes BK,Chalupska D,Gornicki P,Hanna PC,Tsodikov OV,Joachimiak A,Garneau-Tsodikova S

    更新日期:2015-05-26 00:00:00

  • Binding domain of human parathyroid hormone receptor: from conformation to function.

    abstract::A 31 amino acid fragment of the extracellular N-terminus of the human G-protein coupled receptor for parathyroid hormone (PTH1R) has been structurally characterized by NMR and molecular dynamics simulations. The fragment PTH1R[168-198] includes residues 173-189, shown by photoaffinity cross-linking to be a contact dom...


    pub_type: 杂志文章


    authors: Pellegrini M,Bisello A,Rosenblatt M,Chorev M,Mierke DF

    更新日期:1998-09-15 00:00:00

  • Arg352 is a major determinant of charge selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel.

    abstract::The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We inve...


    pub_type: 杂志文章


    authors: Guinamard R,Akabas MH

    更新日期:1999-04-27 00:00:00

  • Affinity labeling of lysine-149 in the anion-binding exosite of human alpha-thrombin with an N alpha-(dinitrofluorobenzyl)hirudin C-terminal peptide.

    abstract::In order to define structural regions in thrombin that interact with hirudin, the N alpha-dinitrofluorobenzyl analogue of an undecapeptide was synthesized corresponding to residues 54-64 of hirudin [GDFEEIPEEY(O35SO3)L (DNFB-[35S]Hir54-64)]. DNFB-[35S]Hir54-64 was reacted at a 10-fold molar excess with human alpha-thr...


    pub_type: 杂志文章


    authors: Bourdon P,Fenton JW 2nd,Maraganore JM

    更新日期:1990-07-10 00:00:00

  • The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates.

    abstract::We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulf...


    pub_type: 杂志文章


    authors: Treadway WJ Jr,Schultz RM

    更新日期:1976-09-21 00:00:00

  • Amyloid formation via supramolecular peptide assemblies.

    abstract::Amyloid fibrils have been classically defined as linear, nonbranched polymeric proteins with a cross beta-sheet structure and the ability to alter the optical properties of the amyloid-specific dye Congo Red. Mounting evidence suggests that soluble oligomeric peptide assemblies approximately 2-20 nm in diameter are cr...


    pub_type: 杂志文章


    authors: Moore RA,Hayes SF,Fischer ER,Priola SA

    更新日期:2007-06-19 00:00:00

  • Pyrene fluorescence analysis offers new insights into the conformation of the lipoprotein-binding domain of human apolipoprotein E.

    abstract::The C-terminal domain (CT) of apolipoprotein E (apoE), a critical protein involved in cholesterol transport in the plasma and brain, plays an important role in high-affinity lipoprotein binding. Although high-resolution structural information is available for the N-terminal domain of apoE, the structural organization ...


    pub_type: 杂志文章


    authors: Patel AB,Khumsupan P,Narayanaswami V

    更新日期:2010-03-02 00:00:00

  • The BARD1 C-terminal domain structure and interactions with polyadenylation factor CstF-50.

    abstract::The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the B...


    pub_type: 杂志文章


    authors: Edwards RA,Lee MS,Tsutakawa SE,Williams RS,Nazeer I,Kleiman FE,Tainer JA,Glover JN

    更新日期:2008-11-04 00:00:00

  • Cyanide binding at the non-heme Fe2+ of the iron-quinone complex of photosystem II: at high concentrations, cyanide converts the Fe2+ from high (S = 2) to low (S = 0) spin.

    abstract::The primary electron acceptor complex of photosystem II, QAFe2+, can bind a number of small molecules at the iron site, including cyanide [Koulougliotis, D., Kostopoulos, T., Petrouleas, V., & Diner, B. A. (1993) Biochim. Biophys. Acta 1141, 275-282)]. In the presence of NaCN (30-300 mM) at pH 6.5, the reduced state, ...


    pub_type: 杂志文章


    authors: Sanakis Y,Petrouleas V,Diner BA

    更新日期:1994-08-23 00:00:00

  • The sigma enigma: in vitro/in silico site-directed mutagenesis studies unveil σ1 receptor ligand binding.

    abstract::The σ1 receptor is an integral membrane protein that shares no homology with other receptor systems, has no unequivocally identified natural ligands, but appears to play critical roles in a wide variety of cell functions. While the number of reports of the possible functions of the σ1 receptor is increasing, almost no...


    pub_type: 杂志文章


    authors: Brune S,Schepmann D,Klempnauer KH,Marson D,Dal Col V,Laurini E,Fermeglia M,Wünsch B,Pricl S

    更新日期:2014-05-13 00:00:00

  • Electron exchange between Fe(II)-horse spleen ferritin and Co(III)/Mn(III) reconstituted horse spleen and Azotobacter vinelandii ferritins.

    abstract::Azotobacter vinelandii bacterioferritin (AvBF) containing 800-1500 Co or Mn atoms as Co(III) and Mn(III) oxyhydroxide cores (Co-AvBF, Mn-AvBF) was synthesized by the same procedure used previously for horse spleen ferritin (HoSF). The kinetics of reduction of Co-AvBF and Mn-AvBF by ascorbic acid are first-order in eac...


    pub_type: 杂志文章


    authors: Zhang B,Harb JN,Davis RC,Choi S,Kim JW,Miller T,Chu SH,Watt GD

    更新日期:2006-05-09 00:00:00

  • Use of pH and kinetic isotope effects to establish chemistry as rate-limiting in oxidation of a peptide substrate by LSD1.

    abstract::The mechanism of oxidation of a peptide substrate by the flavoprotein lysine-specific demethylase (LSD1) has been examined using the effects of pH and isotopic substitution on steady-state and rapid-reaction kinetic parameters. The substrate contained the 21 N-terminal residues of histone H3, with a dimethylated lysyl...


    pub_type: 杂志文章


    authors: Gaweska H,Henderson Pozzi M,Schmidt DM,McCafferty DG,Fitzpatrick PF

    更新日期:2009-06-16 00:00:00

  • Tryptophan residues flanking the second transmembrane helix (TM2) set the signaling state of the Tar chemoreceptor.

    abstract::The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster in patches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellar rotation. The functional domains of a receptor are physically separated by the cell membrane. Chemoeffectors bind to...


    pub_type: 杂志文章


    authors: Draheim RR,Bormans AF,Lai RZ,Manson MD

    更新日期:2005-02-01 00:00:00

  • Urea-induced unfolding of Na,K-ATPase as evaluated by electron paramagnetic resonance spectroscopy.

    abstract::Urea-induced unfolding of Na,K-ATPase from pig kidney and from shark salt gland was studied by electron paramagnetic resonance (EPR) spectroscopy of a nitroxyl derivative of maleimide covalently attached to sulfhydryl groups which are essential for activity. Urea-induced structural changes lead to the inhibition of Na...


    pub_type: 杂志文章


    authors: Babavali M,Esmann M,Fedosova NU,Marsh D

    更新日期:2009-09-29 00:00:00

  • High resolution solution structure of the 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

    abstract::Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxalacetate. Within the multi-subunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier and also binds the ot...


    pub_type: 杂志文章


    authors: Reddy DV,Shenoy BC,Carey PR,Sönnichsen FD

    更新日期:2000-03-14 00:00:00

  • Polymorphism of F-actin assembly. 2. Effects of barbed end capping on F-actin assembly.

    abstract::In the accompanying paper [Suzuki, A., Yamazaki, M., & Ito, T. (1996) Biochemistry 35, 5238-5244], we presented a quantitative phase diagram of actin filament ( (F-actin) described with the F-actin concentration and delta chi value which characterizes the affinity of F-actin with solvent. The phase diagram shows that ...


    pub_type: 杂志文章


    authors: Suzuki A,Ito T

    更新日期:1996-04-23 00:00:00

  • Specificity of the bacteriophage PBS2 induced inhibitor of uracil-DNA glycosylase.

    abstract::The purified PBS2 phage-coded inhibitor of uracil-DNA glycosylase (Ura-DNA glycosylase) from Bacillus subtilis has been tested for its ability to inhibit this enzyme isolated from other prokaryotic and from eukaryotic sources. In addition, the inhibitor has been assayed for its effect on DNA glycosylases specific for ...


    pub_type: 杂志文章


    authors: Karran P,Cone R,Friedberg EC

    更新日期:1981-10-13 00:00:00

  • 1-Aminocyclopropanephosphonate: time-dependent inactivation of 1-aminocyclopropanecarboxylate deaminase and Bacillus stearothermophilus alanine racemase by slow dissociation behavior.

    abstract::1-Aminocyclopropanephosphonate (ACPP) was synthesized, and its effects on the pyridoxal 5'-phosphate linked enzymes 1-aminocyclopropanecarboxylate (ACPC) deaminase from Pseudomonas sp. ACPC and alanine racemase from Bacillus stearothermophilus were studied. ACPP was found to be a potent inhibitor of both enzymes with ...


    pub_type: 杂志文章


    authors: Erion MD,Walsh CT

    更新日期:1987-06-16 00:00:00

  • Some effects of ionophore A23187 on energy utilization and the distribution of cations and anions in mitochondria.

    abstract::The effects of ionophore A23187 on the movements of Ca2+, Mg2+, H+ phosphate, and succinate and its effects on energy utilization by mitochondria have been studied as a function of ionophore concentrations. At a low ratio of the compound to mitochondrial protein, below that required for maximal uncoupling, an apparent...


    pub_type: 杂志文章


    authors: Pfeiffer DR,Hutson SM,Kauffman RF,Lardy HA

    更新日期:1976-06-15 00:00:00

  • Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details.

    abstract::The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three m...


    pub_type: 杂志文章


    authors: Makde RD,Mahajan SK,Kumar V

    更新日期:2007-02-27 00:00:00

  • Spin-label detection of hemoglobin-membrane interaction at physiological pH.

    abstract::The interaction between hemoglobin and the cytoplasmic surface of human erythrocyte membranes at physiological pH was studied by monitoring the electron paramagnetic resonance (EPR) signal of spin-labeled membrane ghosts in hemoglobin solutions of various concentrations. The EPR spectra indicate the existence of a sig...


    pub_type: 杂志文章


    authors: Fung LW

    更新日期:1981-12-08 00:00:00

  • 6-(Difluoromethyl)tryptophan as a probe for substrate activation during the catalysis of tryptophanase.

    abstract::A substrate analogue, 6-(difluoromethyl)tryptophan, was developed and characterized for mechanistic investigation of tryptophanase. The utility of this derivative was based on its ability to partition between fluoride elimination and carbon-carbon bond scission during tryptophan metabolism. The non-enzymatic hydrolysi...


    pub_type: 杂志文章


    authors: Woolridge EM,Rokita SE

    更新日期:1991-02-19 00:00:00