Steen Uttrup Pedersen

Functional mRNA half lives in E. coli

SummaryAnalysis of the synthetic rate of individual protein species at various times after complete inhibition of transcription with either streptolygidin or rifampicin was carried out by two-dimensional polyacrylamide electrophoresis of total Escherichia coli cell extracts. The decay rate of the potential to synthesize different proteins was assumed to be equal to the functional decay rate of the corresponding mRNA. We conclude the following: (a) The tufA and tufB messengers have different half lives (3.0 and 2.4 min, recpectively). (b) Different genes within the same transcriptional unit can have different half lives (S7, EFG and EFTuA — 2.5, 3.8 and 3.0 min, respectively). (c) There is at least a twenty-fold variation in individual mRNA half lives in E. coli; ribosomal protein Sl mRNA was observed to have the shortest half life in the cell (40 sec), while the longest observed half life was approximately 20 min (all values at 30°C). (d) addition of rifampicin increases the absolute rate of RNA polymerase subunit α and β synthesis twofold. (e) The induction of the synthesis of α subunit of RNA polymerase takes palce without a concomitant induction of ribosomal protein S4 and L17, which are reported to be on either side of α in the same transcriptional unit.

On Electron Transfer in Aliphatic Nucleophilic Substitution

Since the classical studies by Ingold in the nineteen thirties the mechanism of the aliphatic nucleophilic substitution reaction has been described as a polar two-electron mechanism in which the nucleophile transfers two electrons to the new bond which is established to the electrophilic center. Depending on the reaction order the abbreviations SN1 and SN2 were used for first-order and second-order reactions, respectively.

New methods for the accurate determination of extinction and diffusion coefficients of aromatic and heteroaromatic radical anions in N,N-dimethylformamide

Abstract Two electrochemical methods are presented that allow an easy access to highly accurate UV-visible spectra of aromatic and heteroaromatic radical anions with life times longer than 50 s in N , N -dimethylformamide. The uncertainty on the extinction coefficients obtained is estimated to be as low as 5% which is a significant improvement in accuracy compared with other methods. The study has involved the radical anions of benzophenone, fluorenone, 1,4-naphthoquinone, anthraquinone, 1,2-benzanthraquinone, (E) -stilbene, anthracene, 9,10-diphenylanthracene, fluoranthene, perylene, benzonitrile, p -tolunitrile, 1,4-diacetylbenzene, 1,4-dicyanobenzene, 9,10-dicyanoanthracene, (E) -azobenzene, ( E )-4-methoxycarbonylazobenzene, quinoxaline and phenazine. The first method takes advantage of a galvanostatic generation of the radical anions from the parent compounds combined with the recording of the absorbance at specific wavelengths by means of a so-called dip probe. The second technique described is based on the continuous and complete electroreduction of the aromatic compounds in an efficient electrochemical flow cell placed just ahead of a quartz cuvette in a traditional diode-array spectrometer. The successful strategy of combining a galvanostatic generation of aromatic radical anions with the measurement of a characteristic physical property has also been employed in the determination of diffusion coefficients. The recording device is here a rotating disc electrode.

Superhydrophilic polyelectrolyte brush layers with imparted anti-icing properties : Effect of counter ions

This work demonstrates the feasibility of superhydrophilic polyelectrolyte brush coatings for anti-icing applications. Five different types of ionic and nonionic polymer brush coatings of 25-100 nm thickness were formed on glass substrates using silane chemistry for surface premodification followed by polymerization via the SI-ATRP route. The cationic [2-(methacryloyloxy)ethyl]trimethylammonium chloride] and the anionic [poly(3-sulfopropyl methacrylate), poly(sodium methacrylate)] polyelectrolyte brushes were further exchanged with H+, Li+, Na+, K+, Ag+, Ca2+, La3+, C16N+, F-, Cl-, BF4-, SO4(2-), and C12SO3- ions. By consecutive measurements of the strength of ice adhesion toward ion-incorporated polymer brushes on glass it was found that Li+ ions reduce ice adhesion by 40% at -18 °C and 70% at -10 °C. Ag+ ions reduce ice adhesion by 80% at -10 °C relative to unmodified glass. In general, superhydrophilic polyelectrolyte brushes exhibit better anti-icing property at -10 °C compared to partially hydrophobic brushes such as poly(methyl methacrylate) and surfactant exchanged polyelectrolyte brushes. The data are interpreted using the concept of a quasi liquid layer (QLL) that is enhanced in the presence of highly hydrated ions at the interface. It is suggested that the ability of ions to coordinate water is directly related to the efficiency of a given anti-icing coating based on the polyelectrolyte brush concept.

Nitrophenyl Groups in Diazonium-Generated Multilayered Films: Which are Electrochemically Responsive?

Various nitrophenyl-containing organic layers have been electrografted to glassy carbon surfaces using diazonium chemistry to elucidate the extent by which the layer structure influences the solvent (i.e., acetonitrile) accessibility, electroactivity, and chemical reactivity of the films. For most of these films, cyclic voltammetric and impedance spectroscopy measurements show that the electron-transfer process at the electrode is facile and independent of film thickness and structure. This is consistent with the occurrence of self-mediated electron transfers throughout the film with nitrophenyl groups serving as redox stations. Importantly, this behavior is seen only after the first potential sweep, the effect of which is to increase the porosity of the layer by inducing an irreversible desorption of nonchemisorbed material along with a reorganization of the film structure. From a kinetic point of view, the radical anions of surface-attached nitrophenyl groups are reactive toward the residual water present in acetonitrile. Thin layers (thickness of 1 to 2 nm) containing redox-active groups only in the outer part of the layer are protonated two to three times as fast as groups located in a more hydrophobic but still solvent-accessible inner layer. Hence, kinetic measurements can detect small differences in the layer environment. Finally, a deconvolution of the cyclic voltammetric response of an electrode grafted from 4-nitrobenzenediazonium discloses that roughly 25% of the overall signal can be attributed to the presence of 4-azonitrophenyl moieties introduced during the electrografting process.

A hybridization assay specific for ribosomal RNA fromEscherichia coli

SummaryRibosomal RNA fromEscherichia coli forms specific hybrids with heterologous DNA fromVibrio metschnicovii. The conditions for such hybridization are described, in which the amount of mRNA fromE. coli binding to theVibrio DNA is about 3% of the amount which hybridizes to the same amount of DNA fromE. coli.

On Surface-Initiated Atom Transfer Radical Polymerization Using Diazonium Chemistry To Introduce the Initiator Layer

This work features the controllability of surface-initiated atom transfer radical polymerization (SI-ATRP) of methyl methacrylate, initiated by a multilayered 2-bromoisobutyryl moiety formed via diazonium chemistry. The thickness as a function of polymerization time has been studied by varying different parameters such as the bromine content of the initiator layer, polarity of reaction medium, ligand type (L), and the ratio of activator (Cu(I)) to deactivator (Cu(II)) in order to ascertain the controllability of the SI-ATRP process. The variation of thickness versus surface concentration of bromine shows a gradual transition from mushroom to brush-type conformation of the surface anchored chains in both polar and nonpolar reaction medium. Interestingly, it is revealed that very thick polymer brushes, on the order of 1 μm, can be obtained at high bromine content of the initiator layer in toluene. The initial polymerization rate and the overall final thickness are higher in the case of nonpolar solvent (toluene) compared to polar medium (acetonitrile or N,N-dimethylformamide). The ligand affects the initial rate of polymerization, which correlates with the redox potentials of the pertinent Cu(II)/Cu(I) complexes (L = Me(6)TREN, PMDETA, and BIPY). It is also observed that the ability of polymer brushes to reinitiate depends on the initial thickness and the solvent used for generating it.

Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1

SummaryTransducing λ phages have been isolated that carry segments of the Escherichia coli chromosome in the aspC region, 20.5 min on the E. coli map. One of these phages λaspC2, carries rpsA, the structural gene for the ribosomal protein S1.A three kilobase fragment from this phage, cloned into either the plasmid pACYC184 or the plasmid pBR322, was found to express S1.In cells carrying the rpsA gene on the high copy number plasmid pBR322 the rate of rpsA mRNA synthesis was increased 40-fold, whereas the rate of protein S1 synthesis was doubled, in comparison with these rates in an rpsA haploid.

Transcriptional organization of the rpsA operon of Escherichia coli.

SummaryThree strong and two minor rpsA promoters were found by nuclease S1 mapping, promoter cloning and in vitro transcription. The longest transcript encodes a protein, located upstream from rpsA with a molecular weight of 25,000. The identity of this protein remains to be established. The other rpsA promoters are located within the gene for this 25 K protein. The rpsA leader region including the sequence of the 25K protein and its promoter was DNA sequenced.

Redox grafting of diazotated anthraquinone as a means of forming thick conducting organic films.

Thick conductive layers containing anthraquinone moieties are covalently immobilized on gold using redox grafting of the diazonium salt of anthraquinone (i.e., 9,10-dioxo-9,10-dihydroanthracene-1-diazonium tetrafluoroborate). This grafting procedure is based on using consecutive voltammetric sweeping and through this exploiting fast electron transfer reactions that are mediated by the anthraquinone redox moieties in the film. The fast film growth, which is followed by infrared reflection absorption spectroscopy, atomic force microscopy, X-ray photoelectron spectroscopy, ellipsometry, and coverage calculation, results in a mushroom-like structure. In addition to varying the number of sweeps, layer thickness control can easily be exerted through appropriate choice of the switching potential and sweep rate. It is shown that the grafting of the diazonium salt is essentially a diffusion-controlled process but also that desorption of physisorbed material during the sweeping process is essentially for avoiding blocking of the film due to clogging of the electrolyte channels in the film. In general, sweep rates higher than 0.5 V s(-1) are required if thick, porous, and conducting films should be formed.