Impaired initiation complex formation on ribosomes treated with colicin E3.

Ribosomes treated with colicin E3 are impaired, when tested with phage R17RNA as messenger, in amino acid incorporation, binding of fMet-tRNA, and formation of fMet-puromycin. Studies with subunits showed that formation of the 30S initiation complex is inhibited. The results are consistent with the recent findings that the “E3-fragment” (3′ end of 16S RNA) binds to 30S protein S1, and that both are involved in initiation on natural messenger.     

Lipid-mediated inactivation of colicin E1 channels by calcium ions

Based on the model of a toroidal protein-lipid pore, the effect of calcium ions on colicin E1 channel was predicted. In electrophysiological experiments Ca2+ suppressed the activity of colicin E1 channels in membranes formed of diphytanoylphosphatidylglycerol, whereas no desorption of the protein occurred from the membrane surface. The effect of Ca2+ was not observed on membranes formed of diphytanoylphosphatidylcholine. Single-channel measurements revealed that Ca2+-induced reduction of the colicin-induced current across the negatively charged membrane was due to a decrease in the number of open colicin channels and not changes in their properties. In line with the toroidal model, the effect of Ca2+ on the colicin E1 channel-forming activity is explained by alteration of the membrane lipid curvature caused by electrostatic interaction of Ca2+ with negatively charged lipid head groups.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

Crystal structure of colicin E3: implications for cell entry and ribosome inactivation.

Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.     

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.     

In vitro depolarization of Escherichia coli membrane vesicles by colicin Ia.

Conditions are reported under which membrane vesicles prepared from Escherichia coli K12 are depolarized by colicin Ia. Although incubation of membrane vesicles with active colicin Ia affects neither transport activity nor the ability of such vesicles to generate a deltapH or deltapsi, a single freeze-thaw cycle of such vesicles in the presence of colicin Ia leads to 1) retention of the colicin by the vesicles, 2) inactivation of transport activity, and 3) membrane depolarization, with a concomitant increase in the transmembrane deltapH. These effects are dependent upon the presence of active colicin Ia during the freeze-thaw cycle. These findings are consistent with our previous results showing that Ia-treated whole cells or membrane vesicles prepared from such cells are defective in their ability to generate a deltapsi, yet generate an increased deltapH (Tokuda, H., and Konisky, J. (1978) Proc. Natl. Acad. Sci. U. S. A., 75, 2579--2583). In addition to its effect on vesicles prepared from sensitive cells, we show that vesicles prepared from both colicin Ia-resistant and -tolerant cells are depolarized by colicin treatment with a concomitant increase in deltapH. It is concluded that the final target of colicin Ia is the cytoplasmic membrane. A model for the mechanism of colicin Ia action is presented in which colicin Ia binds to the specific colicin Ia outer membrane receptor and is subsequently translocated to the cytoplasmic membrane where its integration leads to the formation of ion channels.     

In vitro inactivation of methionine synthase by nitrous oxide

Nitrous oxide (N2O) is commonly used as an anesthetic agent. Prolonged exposure to N2O leads to megaloblastic anemia in humans and to loss of methionine synthase activity in vertebrates. We now report that purified preparations of cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) from both Escherichia coli and pig liver are irreversibly inactivated during turnover in buffers saturated with N2O. Inactivation by N2O occurs only in the presence of all components required for turnover: homocysteine, methyltetrahydrofolate, adenosylmethionine, and a reducing system. Reisolation of the inactivated E. coli enzyme after turnover in the presence of N2O resulted in significant losses of bound cobalamin and of protein as compared to controls where the enzyme was subjected to turnover in N2-equilibrated buffers before reisolation. However, N2O inactivation was not associated with major changes in the visible absorbance spectrum of the remaining enzyme-bound cobalamin. We postulate that N2O acts by one-electron oxidation of the cob(I)alamin form of the enzyme which is generated transiently during turnover with the formation of cob(II)alamin, N2, and hydroxyl radical. Generation of hydroxyl radical at the active site of the enzyme could explain the observed irreversible loss of enzyme activity.     

Mapping of colicin E2 and colicin E3 plasmid deoxyribonucleic acid EcoR-1-sensitive sites.

Colicin plasmids E2 and E3 (Col E2 and Col E3) deoxyribonucleic acid (DNA) has been shown to contain, respectively, two and three EcoR1 restriction endonuclease-sensitive sites. This was determined by measuring the DNA fragments generated after EcoR1 endonuclease treatment by agarose gel electrophoresis and electron microscopy. The structure of heteroduplex Col E2-col E3 DNA molecules formed from EcoR1-generated fragments permitted a localization of the EcoR1-sensitive sites on the plasmid chromosomes.     

Singlet oxygen quenchers and the photodynamic inactivation of E. coli ribosomes by methylene blue

$\text{E. coli}$ ribosomes are readily photoinactivated by methylene blue in the presence of air. A variety of singlet oxygen quenchers like NaN₃, 2,5-dimethylfuran, hydroquinone and ascorbic acid provide about 60% protection against this photoinactivation indicating that a major mechanism of ribosome inactivation proceeds through the formation of singlet oxygen, with small contributions (<40%) from other mechanisms. The singlet oxygen quenchers, 1,4-diazabicyclo [2.2.2] octane and triethylamine give unexpected results, in that they show no protection against photoinactivation.     

In vitro mapping of calnexin interaction with ribosomes

Calnexin is an endoplasmic reticulum (ER) resident type I integral membrane phosphoprotein. This protein is actively involved in the ER glycoprotein quality control through its luminal domain. In addition, although calnexin also interacts with membrane-bound ribosomes, the nature of this interaction remains poorly characterized. Herein, using in vitro approaches, we demonstrate that calnexin cytosolic domain directly interacts with, at least 5 ribosomal proteins. Furthermore, we characterize more specifically its interaction with the ribosomal protein L4 and that L4 binds to the 19 carboxy terminal amino acids of calnexin. We suggest that the direct interaction of calnexin with membrane-bound ribosomes may represent a regulatory mechanism for its lectin-like chaperone function.     

In vitro crystallization of ribosomes from chick embryos

A new two-dimensional ribosome crystal, having the tetragonal space group P42(1)2 (a = 593 A), has been grown from ribosome tetramers extracted from hypothermic chick embryos. It is of particular interest because of its larger size (up to 3 x 3 micron2) and greater stability compared to other related polymorphic forms, and because it can easily be grown in large amounts. X-ray diffraction shows the order in the crystal to extend to a resolution of at least 60 A. The crystalline ribosomes appear to contain a full complement of small and large ribosomal subunit proteins and an additional four proteins not characteristic of chick embryo polysomes.     

In vitro selection of RNA aptamers that bind to colicin E3 and structurally resemble the decoding site of 16S ribosomal RNA

Colicin E3 is a ribonuclease that specifically cleaves at the site after A1493 of 16S rRNA in Escherichia coli ribosomes, thus inactivating translation. To analyze the interaction between colicin E3 and 16S rRNA, we used in vitro selection to isolate RNA ligands (aptamers) that bind to the C-terminal ribonuclease domain of colicin E3, from a degenerate RNA pool. Although the aptamers were not digested by colicin E3, they specifically bound to the protein (K(d) = 2-14 nM) and prevented the 16S rRNA cleavage by the C-terminal ribonuclease domain. Among these aptamers, aptamer F2-1 has a sequence similar to that of the region around the cleavage site from residue 1484 to 1506, including the decoding site, of E. coli 16S rRNA. The secondary structure of aptamer F2-1 was determined by the base pair covariation among the variants obtained by a second in vitro selection, using a doped RNA pool based on the aptamer F2-1 sequence. The sequence and structural similarities between the aptamers and 16S rRNA provide insights into the recognition of colicin E3 by this specific 16S rRNA region.