Regulation of intracellular protein breakdown in stringent and relaxed strains of E. coli,

Regulation of protein breakdown by amino acids is abolished in relaxed strains of E. coli, but these mutants do respond to the deprivation of inorganic phosphate. Protein synthesis is directly required for the control of protein degradation. We suggest that the failure of amino acid-deprived rel- strains to regulate protein breakdown may be due to defective protein synthesis.     

Uptake and aminoacylation of exogenous Escherichia coli tRNA by mouse fibroblasts.

Mouse fibroblasts (L-cells) in suspension culture take up exogenous $\text{Escherichia coli}$ tRNA in the presence of DEAE-Dextran. Tritium-labeled formylmethionine tRNA and valine tRNA are both taken up at very low levels. Tritium activity is associated solely with 4S material as judged by chromatography of cell extracts on Sephadex G-100. Further analysis of this material on a dihydroxyboryl-substituted cellulose indicates that a small portion of the tRNA taken up is acylated by the L-cells.     

Molecular mimicry in translational control of E. coli threonyl-tRNA synthetase gene. Competitive inhibition in tRNA aminoacylation and operator-repressor recognition switch using tRNA identity rules.

We previously showed that: (i) E.coli threonyl-tRNA synthetase (ThrRS) binds to the leader of its mRNA and represses translation by preventing ribosome binding to its loading site; (ii) the translational operator shares sequence and structure similarities with tRNA(Thr); (iii) it is possible to switch the specificity of the translational control from ThrRS to methionyl-tRNA synthetase (MetRS) by changing the CGU anticodon-like sequence to CAU, the tRNA(Met) anticodon. Here, we show that the wild type (CGU) and the mutated (CAU) operators act as competitive inhibitors of tRNA(Thr) and tRNA(fMet) for aminoacylation catalyzed by E.coli ThrRS and MetRS, respectively. The apparent Kd of the MetRS/CAU operator complex is one order magnitude higher than that of the ThrRS/CGU operator complex. Although ThrRS and MetRS shield the anticodon- and acceptor-like domains of their respective operators, the relative contribution of these two domains differs significantly. As in the threonine system, the interaction of MetRS with the CAU operator occludes ribosome binding to its loading site. The present data demonstrate that the anticodon-like sequence is one major determinant for the identity of the operator and the regulation specificity. It further shows that the tRNA-like operator obeys to tRNA identity rules.     

Error propagation in E. coli protein synthesis

A new approach to the error catastrophe theory, proposed by Leslie Orgel, is presented here. Our model is a development of previous models, but differs in several respects: the overall activity is assumed to be dependent on the error level, the effect of errors in the translating system, giving rise to additional errors in the succeeding generation of products, is explicitly included as a special term in our model, and scavenging enzymes are assumed to break down and eliminate products with a loose structure. Their efficiency is dependent on the error level. The model also takes into account the dilution of incorrect ribosomes and enzymes, and is described by a time-dependence in terms of ribosome/enzyme generations. The model and the contribution to the time development are discussed in the light of experiments on E. coli treated with streptomycin.     

Requirement for protein synthesis in the regulation of protein breakdown in cultured hepatoma cells.

The modes of action of insulin and of inhibitors of protein synthesis on the degradation of labeled cellular proteins have been studied in cultured hepatoma (HTC) cells. Protein breakdown is accelerated upon the deprivation of serum (normally present in the culture medium), and this enhancement is inhibited by either insulin or cycloheximide. An exception is a limited class of rapidly turning over cellular proteins, the degradation of which is not influenced by insulin or cycloheximide. Alternative hypotheses to explain the relationship of protein synthesis to the regulation of protein breakdown, viz., control by the levels of precursors of protein synthesis, regulation by the state of the ribosome cycle, or requirement for a product of protein synthesis, have been examined. Protein breakdown was not influenced by amino acid deprivation, and measurements of valyl-tRNA levels in HTC cells subjected to various experimental conditions showed no correlation between the levels of charged tRNAVal and the rates of protein degradation. Three different inhibitors of protein synthesis (puromycin, pactamycin, and cycloheximide) suppressed enhanced protein breakdown in a similar fashion. A direct relationship was found between the respective potencies of these drugs to inhibit protein synthesis and to block enhanced protein breakdown. When cycloheximide and insulin were added following a prior incubation of HTC cells in a serum-free medium, protein breakdown was maximally suppressed within 15-30 min. Actinomycin D inhibited protein breakdown only after a time lag of about 90 min. It is suggested that the regulation of protein breakdown in hepatoma cells requires the continuous formation of a product of protein synthesis, in a manner analogous to the mode of the control of this process in bacteria.     

Stringent control of protein synthesis in E. coli

The effect of ppGpp on the rate of protein synthesis has been determined in vivo. When the stringent response is triggered and then reversed in isogenic strains carrying either spoT^? or spoT⁺, the mutants lower their elevated ppGpp only slowly, whereas wild type cells do so rapidly. Protein synthesis resumes only after a lag in spoT^? cells but almost immediately in spoT⁺ cells. In spoT^?rel^? double mutants, ppGpp does not accumulate under these conditions and protein synthesis resumes immediately. Inhibition of protein synthesis in spoT^?rel⁺ cells therefore appears to be due to elevation of ppGpp levels and not to any other effect of the spoT^? mutation.     

Roles of creatine in the regulation of cardiac protein synthesis

The observation that increased muscular activity leads to muscle hypertrophy is well known, but identification of the biochemical and physiological mechanisms by which this occurs remains an important problem. Experiments have been described (5, 6) which suggest that creatine, an end product of contraction, is involved in the control of contractile protein synthesis in differentiating skeletal muscle cells and may be the chemical signal coupling increased muscular activity and the increased muscular mass. During contraction, the creatine concentration in muscle transiently increases as creatine phosphate is hydrolyzed to regenerate ATP. In isometric contraction in skeletal muscle for example, Edwards and colleagues (3) have found that nearly all of the creatine phosphate is hydrolyzed. In this case, the creatine concentration is increased about twofold, and it is this transient change in creatine concentration which is postulated to lead to increased contractile protein synthesis. If creatine is found in several intracellular compartments, as suggested by Lee and Vissher (7), local changes in concentration may be greater then twofold. A specific effect on contractile protein synthesis seems reasonable in light of the work of Rabinowitz (13) and of Page et al. (11), among others, showing disproportionate accumulation of myofibrillar and mitochondrial proteins in response to work-induced hypertrophy and thyroxin-stimulated growth. Previous experiments (5, 6) have shown that skeletal muscles cells which have differentiated in vitro or in vivo synthesize myosin heavy-chain and actin, the major myofibrillar polypeptides, faster when supplied creatine in vitro. The stimulation is specific for contractile protein synthesis since neither the rate of myosin turnover nor the rates of synthesis of noncontractile protein and DNA are affected by creatine. The experiments reported in this communication were undertaken to test whether creatine selectively stimulates contractile protein synthesis in heart as it does in skeletal muscle.     

Does the regulation of intracellular protein degradation require protein synthesis?

Temperature-sensitive mutants of mammalian cells were used to show that regulation of protein degradation occurs in the absence of protein synthesis.     

The relative importance of muscle protein synthesis and breakdown in the regulation of muscle mass.

The effects of growth-suppressing and muscle-wasting treatments on muscle protein turnover and amino acid concentrations were determined in vivo. All treatments depressed protein synthesis and some treatments depressed protein breakdown. Only prolonged starvation increased protein breakdown. Muscle protein mass is regulated primarily through alterations in protein synthesis in all except emergency conditions. The increased concentrations of the branched-chain amino acids indicate that they are unlikely to be involved in this regulation.     

Base substitutions in the wobble position of the anticodon inhibit aminoacylation of E. coli tRNAfMet by E. coli Met-tRNA synthetase

Derivatives of E. coli tRNAfMet containing single base substitutions at the wobble position of the anticodon have been enzymatically synthesized in vitro. The procedure involves excision of the normal anticodon, CAU, by limited digestion of intact tRNAfMet with RNase A. RNA ligase is then used to join each of four trinucleotides, NAU, to the 5' half molecule and to subsequently link the 3' and modified 5' fragments to regenerate the anticodon loop. Synthesis of intact tRNAfMet containing the anticodon CAU by this procedure yields a product which is indistinguishable from native tRNAfMet with respect to its ability to be aminoacylated by E. coli methionyl-tRNA synthetase. Substitution of any other nucleotide at the wobble position of tRNAfMet drastically impairs the ability of the synthetase to recognize the tRNA. Measurement of methionine acceptance in the presence of high concentrations of pure enzyme has established that the rate of aminoacylation of the AAU, GAU and UAU anticodon derivatives of tRNAfMet is four to five orders of magnitude slower than that of the native or synthesized tRNA containing C as the wobble base. In addition, the inactive tRNA derivatives fail to inhibit aminoacylation of normal tRNAfMet, indicating that they bind poorly to the enzyme. These results support a model involving direct interaction between Met-tRNA synthetase and the C in the wobble position during aminoacylation of tRNAfMet.     

Aging changes in intracellular protein breakdown

Using radioactively labelled cytosol proteins as substrates we were able to exclude the possible accumulation of any specific inhibitor for the lysosomal proteases in rat liver cytosol during the aging process. There were also no gross changes in the molecular weight patterns of these proteins during the aging process. The percentage of more hydrophobic proteins seems to be identical in both the "old" and "young" cytosol proteins. From immunological experiments we suppose a qualitative change in the composition of rat liver cytosol proteins or of their properties during the aging process.     

Efficiency of phage protein synthesis in lambda-infected E. coli minicells

Phage lambda major head protein, the gene E product, has been identified among other phage proteins synthesized in lambda-infected Escherichia coli minicells, separated by SDS-acrylamide gel electrophoresis. On stained gels, the same protein has also been detected among total (bacterial and phage) proteins of lambda-infected minicells. The contribution of lambda proteins to the total protein content of lambda-infected minicells was found to be about 12% following 30 min lambda-infection. The inhibition of lambda early protein synthesis (shown by other authors in nucleate bacterial cells) practically does not occur in minicells; this may be the reason of the observed high efficiency of phage protein synthesis.     

ATP-induced activation of the aminoacylation of tRNA by the isoleucyl-tRNA synthetase from Escherichia coli

The rate of aminoacylation of tRNA catalyzed by the isoleucyl-tRNA synthetase form Escherichia coli has been measured. A steady-state kinetic analysis of the rate as a function of the concentration of ATP gave nonlinear Hanes plots. ATP behaves as an activator of the reaction. The activation is observed at a low magnesium ion concentration and in the presence of spermidine. The presence of inorganic pyrophosphate or AMP enhances the activation. The results are consistent with a mechanism in which the binding of a second molecule of ATP increases the rate of dissociation of Ile-tRNA from the enzyme.     

E. coli proteolytic activity in milk and casein breakdown

Previous studies have focused on both LPS and E. coli experimental mastitis and underlined the respective roles of endogenous proteolysis (including plasmin from the blood stream and other proteases from milk leukocytes), as well as the presence of E. coli in a more intricate system. The aim of this study was to assess the role of E. coli in milk proteolysis and especially that of its proteases in casein breakdown. The first part consisted in the incubation of 104 cfu.mL(-1) of the E. coli strain in raw milk at 37 degrees C for 24 h; the same milk was also incubated with 0.04% sodium azide. Several parameters were evaluated: CFU, plasmin activity, gelatinase activity and pH 4.6 insoluble peptides, including the proportion of gamma-CN. The profile of gelatinase activity was determined by zymography and identified by immunoblotting. In the second part of the study, we examined the profile of CN (alphas-, beta- and kappa-CN) breakdown by E. coli lysate. The results suggest that E. coli proteases have a direct effect on CN, and the increase of gamma-CN in inoculated milk may be generated by both plasmin and the gelatinase. Moreover, the gelatinase activity in the inoculated milk was higher after 24 h of incubation.