Highly selective labeling of the E. coli RNA-polymerase promoter complex with reactive derivatives of oligonucleotide primers of various specificity

A technique of highly selective affinity labelling, which includes covalent modification of the enzyme-T7A2 promoter complex with reactive oligonucleotide derivatives and subsequent elongation of the attached oligonucleotide residue with a radioactive substrate was used to study the product-binding site of E. coli RNA polymerase. Different oligonucleotides complementary to the T7A2 promoter (with lengths ranging from 2 to 8 residues) containing 5'-terminal phosphorylating, alkylating or aldehyde groups were used for the labelling. The procedure resulted in labelling DNA and beta-, beta'- or sigma-subunits of the enzyme, which are therefore believed to contact with growing RNA in the course of initiation. Consideration of the labelling patterns as a functions of the oligonucleotide's length as well as of the structure and chemical specificity of the reactive groups led to a tentative topographic scheme of the RNA polymerase product-binding region.     

Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1

Abstract Pulse labelling experiments and enzyme studies show that Thermoproteus tenax is able to degrade glucose via the Embden-Meyerhof-Parnas (EMP) pathway. T. tenax is the first archeum for which the glycolytic EMP pathway could be established. One of the key enzymes, 6-phosphofructokinase of T. tenax depends on (pyrophosphate-fructose-6-phosphate-1-phosphotransferase) instead of ATP as found in some bacterial and eucaryal species. In addition to the intermediates of the EMP pathway the intermediary products of the non-phosphorylated Entner-Doudoroff pathway were also detected by pulse labelling experiments indicating that under chemoorganotrophic conditions at least 2 glycolytic pathways are operative in T. tenax .     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Solubilization of peroxidase from porcine thyroids and characterization by photoaffinity labelling

Peroxidase was solubilized without proteolysis from porcine thyroid particulate fraction with the nonionic detergent, 1-O-n-octyl-beta-D-glucopyranoside. The enzyme was able to catalyze the oxidation of guaiacol and the iodination of bovine serum albumin (33 atoms of iodine per molecule protein). Binding studies performed with the partially purified enzyme indicated that the substrates thyroxine (T4) and tyrosine compete for the same binding site on the enzyme. Dissociation constants of 0.9 nM and 0.5 nM were found for T4 and tyrosine, respectively. After photoaffinity labelling with underivatized 125I-labelled T4, gel chromatography on Sephacryl S-1000 revealed a relative molecular weight of about 100 000 for the solubilized enzyme. The peroxidase activity and haem-absorbance peak coeluted from the Sephacryl S-1000 column. SDS-polyacrylamide gel electrophoresis under reducing conditions indicated two major radiolabelled polypeptides, Mr 83 000 and Mr 42 600, as well as a smaller peak at Mr 15 400. The 15 400 molecular weight species is probably not part of the peroxidase complex, since it could partially be removed by Sephadex G-25 prechromatography . Further analyses confirmed that the partially purified enzyme is a haemoprotein absorbing maximally at 412 nm. The Soret band is shifted to 423 nm by reducing agents and the haem-cyanide complex has a maximum absorbance at 416 nm.     

Affinity labelling of alcohol dehydrogenases. Chemical modification of the horse liver and the yeast enzymes with alpha-bromo-beta(5-imidazolyl)-propionic acid and 1,3-dibromoacetone.

1. DL-alpha-Bromo-beta(5-imidazolyl)-propionic acid is a potential affinity labelling reagent for metallo-enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis-Menten-type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2'-dipyridyl, 1,10-phenanthroline and cyclohexanone, which all combine with the active-site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys-46, reversible binding is shown to occur at the active-site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found. 2. The potential affinity labelling and crosslinking reagent, symmetrical 1,3-dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis-Menten-type at pH 7.0. Several ligands known to complex with the active-site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo-first-order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.     

Interpretation of the rate of density labelling of enzymes with 2H2O. Possible implications for the mode of action of phytochrome.

The interpretation of the rate of density labelling of enzymes (the differential or comparative density labelling technique) is considered in terms of two simple models of enzyme turnover. On the basis of this illustrative analysis it is demonstrated that the interpretation of differential (comparative) density labelling experiments is critically dependent on the establishment of a definitive mechanism of enzyme turnover for each particular enzyme studied. In the light of these findings the interpretation of comparative (differential) density labelling experiments appertaining to the mechanism of phytochrome-mediated increases in enzyme activity in higher plants is re-assessed.     

Thermal and urea-induced unfolding in T7 RNA polymerase: calorimetry, circular dichroism and fluorescence study

Structural changes in T7 RNA polymerase (T7RNAP) induced by temperature and urea have been studied over a wide range of conditions to obtain information about the structural organization and the stability of the enzyme. T7RNAP is a large monomeric enzyme (99 kD). Calorimetric studies of the thermal transitions in T7RNAP show that the enzyme consists of three cooperative units that may be regarded as structural domains. Interactions between these structural domains and their stability strongly depend on solvent conditions. The unfolding of T7RNAP under different solvent conditions induces a highly stable intermediate state that lacks specific tertiary interactions, contains a significant amount of residual secondary structure, and undergoes further cooperative unfolding at high urea concentrations. Circular dichroism (CD) studies show that thermal unfolding leads to an intermediate state that has increased beta-sheet and reduced alpha-helix content relative to the native state. Urea-induced unfolding at 25 degrees C reveals a two-step process. The first transition centered near 3 M urea leads to a plateau from 3.5 to 5.0 M urea, followed by a second transition centered near 6.5 M urea. The CD spectrum of the enzyme in the plateau region, which is similar to that of the enzyme thermally unfolded in the absence of urea, shows little temperature dependence from 15 degrees to 60 degrees C. The second transition leads to a mixture of poly(Pro)II and unordered conformations. As the temperature increases, the ellipticity at 222 nm becomes more negative because of conversion of poly(Pro)II to the unordered conformation. Near-ultraviolet CD spectra at 25 degrees C at varying concentrations of urea are consistent with this picture. Both thermal and urea denaturation are irreversible, presumably because of processes that follow unfolding.     

Affinity modification of E. coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of primer oligonucleotides

Oligonucleotides 2 to 7 nucleotide residues long, complementary to the codogenic strand of T7 promoter A2, have been synthesized; all of them contained a ribo-unit at the 3'-end. They were converted into 5'-(N-methyl)phosphoimidazolides, and the affinity reagents obtained were allowed to bind covalently to RNA polymerase in the presence of a promoter. Some of the nucleotide residues covalently attached occupied proper positions relative to the active centre of the phosphodiester bond synthesis and on addition of [alpha-32P]UTP were elongated, so that highly selective affinity labelling occurred. With oligonucleotides of various lengths, different distribution of the label between beta, beta' and sigma subunits of RNA polymerase took place. Most efficient was labelling of beta-subunit by the residue--pCpGpCpU, and of sigma-subunit by the residue--pApApApTp-CpGpCpU (p--radioactive phosphorus atom). In both cases, the amino acid residues labelled were histidines.     

Protein kinase of bacteriophage T7. 2. Properties, enzyme synthesis in vitro and regulation of enzyme synthesis and activity in vivo.

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3':5'-AMP and/or cyclic 3':5'-GMP. The T7 protein kinase carries -- SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested.     

DNA filter retention assay for exonuclease activities. Application to the analysis of processivity of phage T5 induced 5'-exonuclease

The 5'-exonuclease of phage T5 has been purified nearly to homogeneity by using a simple and fast procedure. The kinetic properties of the purified enzyme have been studied by using a new sensitive assay based upon retention by nitrocellulose filters of DNA with short protruding single-stranded ends. The enzyme is specifically stimulated by KCl. Its Km is 2.2 X 10(-7) M at 30 degrees C, and its turnover number is 0.33 DNA molecule transformed per minute. The filter retention assay shows that the T5 exonuclease acts by a semiprocessive mechanism, removing from DNA ends about 30 nucleotides on the average per cycle. The degree of enzyme processivity increases with increasing magnesium concentrations.     

The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli.

The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized [125I]di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination. The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments. Hydrogenase (mol.wt. approx. 63 000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.     

Ultrastructural localization of cathepsin B in gingival tissue from chronic periodontitis patients

Summary Loss of tooth support during chronic periodontitis is very likely to involve tissue proteases such as cathepsin B. The distribution of this enzyme was, therefore, examined in ultrathin sections of gingival tissue embedded in acrylic resin and labelled with a sheep polyclonal antibody and gold-conjugated secondary antibody. Macrophages and fibroblasts in both inflamed and non-inflamed areas of tissue showed labelling, and this was strongest in lysosomes, corresponding to the normal intracellular location of cathepsin B. However, additional gold particles were found on the surface of these cells. Monocytes in inflamed areas also had surface labelling, some of which was present on microvilli. Labelled collagen fibres adjacent to all three cell types indicated that cathepsin B had been released into the immediate extracellular environment. Plasma membrane cathepsin B has previously been associated with cancers, but enzyme redistribution and release in the gingiva may have been linked to the inflammatory response, since fibroblasts and macrophages in non-inflamed areas showed less labelling of their surface and adjacent collagen. The collagen labelling added to evidence that cathepsin B can function extracellularly as well as intracellularly in connective tissue degradation. This destructive role for the enzyme is supported by our earlier measurements of increased biochemical activity in chronic periodontitis This revised version was published online in November 2006 with corrections to the Cover Date.     

Action of Single-strand-specific S1 Endonuclease on Locally Altered Structures in Double-stranded DNA

The cleavage of double-stranded DNA by S1 endonuclease was studied by sucrose density gradient centrifugation analysis. The enzyme introduced no single-strand breaks into native T7 DNA under conditions where heat-denatured T7 DNA was completely degraded. By using enzyme at about 6 times higher the amount required for complete degradation of the heat-denatured DNA, it was possible to make a few single-strand breaks in native T7 DNA. Under the conditions where native T7 DNA is absolutely resistant to the enzyme, the susceptibility of locally altered structures naturally present and/or artificially induced in native double-stranded DNA to the enzyme was studied. It was evidenced that S1 endonuclease can cleave circular covalently closed, superhelical fl RFI DNA, depurinated T7 DNA, bleomycin-treated T7 DNA containing internal single-strand breaks, but not cleave intercalating drug-bound T7 DNA.     

Microinjection of T4 endonuclease V produced by a synthetic denV gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells

A structural gene for T4 endonuclease V was constructed by ligating synthetic oligonucleotides. The endonuclease V was overproduced in E. coli under control of the E. coli tryptophan promoter and purified to apparent homogeneity. The product had comparable DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease activities to the natural enzyme in vitro. When this endonuclease V was microinjected into the cytoplasm of xeroderma pigmentosum (XP) cells of complementation group A, B, C, D, F, G or H, unscheduled DNA synthesis (UDS) above the residual level was detected in all the cells at a dose of about 10(3) molecules following UV irradiation. The gain numbers of UDS in these XP cells increased with increase in the dose of enzyme and reached a plateau at the normal cell level on introduction of about 10(4) molecules. Introduction of more enzyme into either XP cells or normal human cells did not increase the grain number under regular labelling conditions (2.5 h, 37 degrees C). In normal mouse cells, introduction of the enzyme increased the grain number more than 4-fold under the same conditions during at least 8.5 h following UV irradiation. Furthermore, with a labelling time of 30 min, the enzyme more than doubled the grain number even in normal human cells.     

Studies of the binding of Escherichia coli RNA polymerase to DNA. V. T7 RNA chain initiation by enzyme-DNA complexes

Initiation of T7 RNA chains by Escherichia coli RNA polymerase-T7 DNA complexes has been followed using incorporation of λ-³²P-labeled ATP and GTP to determine the relation between the enzyme binding sites and RNA chain initiation sites on the T7 genome. If the period of RNA synthesis is limited to less than two minutes, the stoichiometry of RNA chain initiation can be measured in the absence of chain termination and re-initiation. About 70% of the RNA polymerase holoenzyme molecules in current enzyme preparations are able to rapidly initiate a T7 RNA chain. The ratio of ATP- to GTP-initiated T7 RNA chains is not altered by variations in the number of enzyme molecules added per DNA, nor by alterations in the ionic conditions employed for RNA synthesis. This suggests that RNA chain initiation sites are chosen randomly through binding of RNA polymerase to tight (class A) binding sites on T7 DNA.     

Cloning, expression, and purification of gene 3 endonuclease from bacteriophage T7

The structural gene for the single-stranded endonuclease coded for by gene 3 of bacteriophage T7 has been cloned in pGW7, a derivative of the plasmid pBR322, which contains the lambda PL promoter and the gene for the temperature-sensitive lambda repressor, cI857. The complete gene 3 DNA sequence has been placed downstream of the PL promoter, and the endonuclease is overproduced by temperature induction at mid-log phase of Escherichia coli carrying the recombinant plasmid pTP2. Despite the fact that cell growth rapidly declines due to toxic effects of the excess endonuclease, significant amounts of the enzyme can be isolated in nearly homogeneous form from the induced cells. An assay of nuclease activity has been devised using gel electrophoresis of the product DNA fragments from DNA substrates. These assays show the enzyme to have an absolute requirement for Mg(II) (10 mM), a broad pH optimum near pH 7, but significant activity from pH 3 to pH 9, and a 10-100-fold preference for single-stranded DNA (ssDNA). The enzyme is readily inactivated by ethylenediaminetetraacetic acid or high salt. The differential activity in favor of ssDNA can be exploited to map small single-stranded regions in double-stranded DNAs as shown by cleavage of the melted region of an open complex of T7 RNA polymerase and its promoter.