Shlomo Avital

A convenient procedure for preparing transfer ribonucleic acid from Escherichia coli

Abstract A procedure is described for the isolation of transfer RNA (tRNA) from Escherichia coli . The method is a combination of several known techniques. tRNA is extracted from whole cells with phenol, stripped of attached amino acids at pH 8, preferentially solubilized with 2 M LiCl, and reprecipitated with (NH 4 ) 2 SO 4 . The key step is the use of 2 M LiCl, which separates ribosomal RNA from tRNA rapidly and completely. The method is simple and yields tRNA as active as the best preparations that we have obtained with other methods.

Properties of the catalytic (αβ)-core complex of chloroplast CF1-ATPase

Abstract The CF1(αβ)-core complex previously isolated from the spinach chloroplast CF0F1-ATP synthase contains equal amounts of CF1α- and β-subunits, functions as a soluble Mg2+-ATPase and forms a hybrid F0F1-ATPase when incorporated into β-less Rhodospirillum rubrum membrane-bound F0F1 (Avital, S. and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067–7072). Here, we demonstrate that this soluble spinach CF1(αβ)-Mg+-ATPase, unlike its latent parent CF1-ATPase, does not respond to activation by octyl glucoside, is only slightly stimulated by sulfite and not inhibited by free Mg2+, azide or tentoxin. The CF1(αβ)-ATPase does however bind tentoxin rather tightly and is stimulated by it at concentrations that inhibit the parent CF1-ATPase. Unlike this soluble CF1(αβ)-ATPase, the hybrid Mg2+-ATPase formed by incorporation of the same CF1(αβ) preparation into β-less R. rubrum F0F1, is markedly stimulated by sulfite and completely inhibited by azide and tentoxin. These results indicate that (a), for stimulation by tentoxin the presence of α- and β-subunits from a sensitive CF1 is enough, whereas for inhibition by it other F1-subunits are required and these can be come from a tentoxin resistant F1-species, such as R. rubrum and (b), although the single-copy subunits are not required for F1-ATPase activity, their presence results in increased rates and large changes in the catalytic properties.

Long range RNA-RNA interactions in the 30 S ribosomal subunit of E. coli.

We have attempted to identify long-range interactions in the tertiary structure of RNA in the E. coli 30 S ribosome. Native subunits were cleaved with ribonuclease and separated into nucleoprotein fragments which were deproteinized and fractionated into multi-oligonucleotide complexes under conditions intended to preserve RNA-RNA interactions. The final products were denatured by urea and heat and their constituent oligonucleotides resolved and sequenced. Many complexes contained complementary sequences known to be bound together in the RNA secondary structure, attesting to the validity of the technique. Other co-migrating oligonucleotides, not joined in the secondary structure, contained mutually complementary sequences in locations that allow base-pairing interaction without disrupting pre-existing secondary structure. In seven instances the complementary relationship was found to have been preserved during phylogenetic diversification.

A large nucleoprotein fragment of the 50-S ribosomal subunit of Escherichia coli

A large nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 50-S ribosomes and purified to gel electrophoretic homogeneity. Conditions were employed under which the fragmentation pattern was reproducible and the various fragment fractions were stable and maintained their sedimentation and electrophoretic properties throughout the several preparative and analytical procedures used. Fractions that appeared homogeneous in sucrose gradient centrifugation were found to be heterogeneous by gel electrophoresis. The large fragment was purified to homogeneity by preparative gel electrophoresis. It contained 21 proteins, the 5-S RNA, and two large oligonucleotides which together total about two thirds the molecular weight of the 23-S RNA. Because it can be prepared reproducibly in large quantities and because of its size and stability, the fragment appears suitable for functional and structural studies and as the starting material for further fractionation. An important contributing factor to the observed stability and reproducibility was the maintenance of an unchanging ionic environment. A single buffer was employed throughout all the procedures, and the fragments produced by nuclease digestion were dissociated from each other by heat rather than by changing the medium.

Quenching of Chlorophyl Fluorescence by Carotenoids in a Micellar Model System

In reference to the hypothesis that zeaxanthin is an effective quencher of chlorophyll-a singlet excited state in-vivo, under conditions of “non-photochemical quenching” (Owens 1994 [1]) we performed experiments on the quenching ability of certain carotenoids in an in-vitro micellar model system — 0.4% v/v triton X-100 micelles in formamide/water, 3:1 v/v (Scherz et al. 1985 [2]). The molar concentration of the micelles was estimated by fluorescence intensity measurements of incorporated chlorophyll-a, which exhibited a strong self- quenching effect, at different concentrations, comparing the results to a theoretical model based on random (Poisson) distribution of chlorophyll molecules into the micelles. Addition of carotenoids into the solution containing 0.059 μM micelles and 0.03 μM chlorophyll caused various degrees of chlorophyll fluorescence quenching: β-carotene was the most effective, zeaxanthin was moderate and violaxanthin was a weak quencher. Lutein and neoxanthin enhanced the fluorescence, probably because of intrinsic chlorophyll content which could not be separated out. The results are consistent with the hypothesis that when the number of conjugated double bonds increases to 11 the first excited state of the carotenoid may become an energy acceptor to excited singlet chlorophyll.

Isolation of an Active β Subunit from Chloroplast CFoF1-ATP Synthase

A number of active β-subunits have been isolated from various F1-ATPases after their complete dissociation into individual subunits (1–3). Two of these isolated β-subunits, the E. coli F1-β subunit(Ecβ) and the chloroplast CF1-β subunit (CF1 β), were shown to form hybrid FoF1-complexes upon their reconstitution into β-less R. rubrum chromatophores (3,4). These hybrid complexes restored a high Mg-ATPase activity, but very little ATP synthesis, in the inactive β-less chromatophores. On the other hand, reconstitution of the native R. rubrum F1-β subunit (Rrβ), that has been removed from the chromatophore membrane-bound FoF1-ATP synthase by extraction with LiCl (5,6) led to full restoration of both coupled ATP synthesis and hydrolysis by the β-less chromatophores.