Feedback regulation of ribosomal protein synthesis in E. coli: Localization of the mRNA target sites for repressor action of ribosomal protein L1

E. coli ribosomal protein L1 is a translational repressor of the synthesis in vitro of both proteins encoded in the L11 operon (L11 and L1). L1 is shown to act at a single target site within the first 160 bases of the bicistronic mRNA, near (or at) the translation initiation site of the L11 cistron. Synthesis of L1 apparently requires translation of the preceding L11 cistron, allowing regulation of the synthesis of both proteins from a single mRNA target site. This observation suggests a sequential translation mechanism that results in the equimolar synthesis rates of the two proteins observed in vivo. It was found that the presence of 23S rRNA, but not 16S rRNA, relieves translational inhibition by L1. L1 presumably recognizes structural features of the mRNA target site that are homologous to the L1-binding site of 23S rRNA. Although previous work indicated that translationally inhibited ribosomal protein mRNA is degraded in vivo, L1 repressor action in the present in vitro system was found not to involve mRNA degradation.     

Autoradiographic localization of α-bungarotoxin-binding sites in the carotid body of the rat

Summary Radioiodinated -bungarotoxin (-Bgt) was used to localize -Bgt-acetylcholine receptors in the carotid body of the rat. The gamma spectrometer analyses indicated a high uptake of [¹²⁵I] -Bgt in carotid bodies incubated in vitro (1.51 fmole per organ). Incorporation of the isotope was effectively blocked by pretreatment of carotid bodies with d-tubocurarine and unlabeled -Bgt, but not by atropine. Light microscopic autoradiography showed a heavy labeling of some parenchymal cells. Electron-microscopic autoradiography revealed that labeling was localized along the interface between parenchymal cells, especially where their cytoplasmic processes engage in complex interdigitations. The silver grain counts on electron-microscopic autoradiographs suggest that labelings are preferentially associated with the plasma membrane of certain Type I cells. It is suggested that these Type I cells in the rat's carotid body probably are provided with nicotinic acetylcholine receptors on their plasma membranes.This research was partly supported by a grant from the Edward G. Schleider Foundation of New Orleans.The authors extend appreciation to Drs. Akira Arimura, Dept. of Medicine, for supplying a part of the radioisotope and to James Fisher, Dept. of Pharmacology, Tulane Medical School, for use of the gamma spectrometer. Appreciation also is extended to Mrs. Lia Pedroza for technical assistance     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.     

Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.     

Acetylene reduction with physiological electron donors by extracts and particulate fractions from nitrogen-fixing Azotobacter chroococcum

1. 1. Preparations were obtained from Azotobacter chroococcum which reduced acetylene to ethylene using physiological electron donors instead of sodium dithionite. These preparations fell into two categories: those which required catalytic amounts of benzyl viologen for acetylene reduction and those that did not.

2. 2. Acetylene reduction without benzyl viologen or sodium dithionite was observed only with particles that sedimented at 40 000 × g after disrupting bacteria in the French press or with preparations obtained by disrupting bacteria protected by a mixture of defatted bovine serum albumin-Ficoll-MgCl₂ with liquid N₂; supernatant fractions required benzyl viologen for acetylene reduction.

3. 3. Added ATP inhibited acetylene reduction by large particles; ATP and MgCl₂ were necessary for maximum acetylene reduction with bovine serum albumin-protected preparations.

4. 4. NADH and carbon substrates acted as electron donors but H₂ did not; NAD⁺ was necessary for maximum acetylene reduction with carbon substrates.

5. 5. Anaerobic conditions were necessary for maximum acetylene reduction in all cases.

Woodland Restoration in the Western Australian Wheatbelt: A Conceptual Framework Using a State and Transition Model

Woodlands dominated by Eucalyptus spp. in temperate southeastern and southwestern Australia have been extensively cleared for agriculture and are often badly degraded by livestock grazing. This has resulted in the loss of biodiversity and widespread land degradation. The continuing decline of these woodlands has become a concern for the conservation of biodiversity, and there is a growing interest among farmers, land managers, and researchers in developing techniques for restoring them. Currently few scientific guidelines exist for undertaking woodland restoration programs. We use a state and transition model to develop hypotheses on restoration strategies for salmon gum (Eucalyptus salmonophloia) woodlands. We consider that this approach provides a suitable framework for organizing knowledge and identifying areas where further information is needed, and hence provides a useful starting point for a restoration program. The model has the potential to provide a tool for land managers with which they can assess the action and effort needed to undertake woodland restoration in agricultural landscapes.     

Ganglioside GM1 Enhances Induction by Nerve Growth Factor of a Putative Dimer of TrkA

Abstract: GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6–24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.