Initial Nucleotide Frequencies of Bacterial RNA synthesized during Amino-acid Starvation or Changes of Carbon Source

THERE is growing evidence to indicate that RNA synthesis in bacteria is regulated through adjustment of the frequency of initiation of new RNA molecules. In this framework an understanding of the process of initiation of RNA synthesis takes on a special importance and for this reason we have investigated in varying conditions the composition of the 5′ terminal or first-inserted nucleotide. It has been previously shown that such initiations do not occur randomly, either in vivo¹ or in the proper conditions in vitro^2,3, but that RNA chains are exclusively initiated with the purines, adenosine and guanosine. Additionally, there was a recent suggestion based on in vitro studies that the nucleotide guanosine-3′-diphosphate-5′-diphosphate (MS1), proposed to be a regulatory agent in RNA synthesis, functioned by specifically depressing the frequency of initiation of a large fraction of RNA molecules beginning with guanosine⁴. Here we report, however, that in vivo, in conditions in which regulation of RNA synthesis is manifest, the ratio of molecules initiated with adenosine and guanosine is not changed.     

Uptake of Inorganic Phosphate by Suspension-Cultured Tobacco Cells: Kinetics and Regulation by Pi Starvation

The kinetics of P_i uptake by phosphate-starved and non-starvedtobacco cells (Nicotiana tabacum BY-2) suspension culture wasinvestigated. The kinetic parameters of P_i uptake were determinedby computer simulation of the curve that represented the time-dependentloss of P_i from the culture medium. The uptake profile couldbe completely explained by assuming the existence of only onekind of Michaelis-Menten-type P_i-transport system with an affinityfor P_i (K_m) of about 2.5 µM (the lowest value reportedto date) in both P_i-starved and non-starved cells. No evidencewas obtained suggesting the existence of a "low-affinity" P_i-uptakesystem that has been postulated to exist in several other plantmaterials. The V_max for uptake of P_i by non-starved cells was12 nmol per minute per milliliter of packed cell. Phosphatestarvation increased the V_max more than 5-fold, while it hadno effect on the affinity for P_i. V_max began to increase (atan almost constant rate) just after loss of all P_i from theculture medium and it reached a maximum about 16 hours later.This induction process was completely prevented by the additionof cycloheximide to the culture medium. All these results suggestthat P_i starvation increases the synthesis of a phosphate-carriercomplex that is postulated to be involved in the P_i-uptake process. (Received August 12, 1994; Accepted December 26, 1994)     

Insulin and Amino-acid Regulation of Polysomes in Perfused,Diabetic Rat Liver

IN normal mammalian liver, protein synthesis and concomitant polyribosome assembly are controlled by amino-acids^1–4. A similar control may be exerted by insulin in liver⁵ and muscle⁶. Both the effect of amino-acids on liver and that of insulin on muscle can be demonstrated with isolated, perfused tissue^4,7, but the interrelationship between these agents has not been clarified either in liver or in muscle. We have used perfused livers from diabetic rats to establish whether amino-acids and insulin promote polysomal assembly independently or in concert.     

Regulation of protein synthesis by mRNA structure

In addition to the m⁷G cap structure, the length of the 5 UTR and the position and context of the AUG initiator codon (which have been discussed elsewhere in this volume), higher order structures within mRNA represent a critical parameter for translation. The role of RNA structure in translation initiation will be considered primarily, although structural elements have also been found to affect translation elongation and termination. We will first describe the different effects of higher order RNA structuresper se, and then consider specific examples of RNA structural elements which control translation initiation by providing binding sites for regulatory proteins.     

Degradation of Glycinebetaine by Betaine-Homocysteine Methyltransferase in Aphanothece halophytica: Effect of Salt Downshock and Starvation

We have investigated conditions leading to the degradation of glycinebetaine in Aphanothece halophytica and have shown the activity of betaine-homocysteine methyltransferase (BHMT). The intracellular glycinebetaine level was decreased approximately 50% after 36 h salt downshock from 2.0 m NaCl medium to 0.5 m NaCl medium. A slight additional decrease of glycinebetaine occurred when salt downshock was combined with dark treatment. The omission of carbon and nitrogen sources in the growth medium further decreased intracellular glycinebetaine. The activity of BHMT increased from 0 to 460 nmol h⁻¹mg⁻¹ after 3 h salt downshock. Higher strength of salt downshock resulted in higher activity of the enzyme. Small increase of the enzyme activity was also observed when A. halophytica was deprived of carbon and nitrogen sources in the growth medium. Received: 17 March 2000 / Accepted: 24 April 2000     

Nutritional Regulation of Lignin Degradation by Phanerochaete chrysosporium

Previous studies have shown that a lignin-degrading system appears in cultures of the white rot fungus Phanerochaete chrysosporium in response to nitrogen starvation, apparently as part of secondary metabolism. We examined the influence of limiting carbohydrate, sulfur, or phosphorus and the effect of varying the concentrations of four trace metals, Ca, and Mg. Limitation of carbohydrate or sulfur but not limitation of phosphorus triggered ligninolytic activity. When only carbohydrate was limiting, supplementary carbohydrate caused a transient repression of activity. In carbohydrate-limited cultures, ligninolytic activity appeared when the supplied carbohydrate was depleted, and this activity was associated with a decrease in mycelial dry weight. The amount of lignin degraded depended on the amount of carbohydrate provided, which determined the amount of mycelium produced during primary growth. Carbohydrate-limited cultures synthesized only small amounts of the secondary metabolite veratryl alcohol compared with nitrogen-limited cultures. l-Glutamate sharply repressed ligninolytic activity in carbohydrate-starved cultures, but NH(4) did not. Ligninolytic activity was also triggered in cultures supplied with 37 muM sulfur as the only limiting nutrient. The balance of trace metals, Mg, and Ca was important for lignin degradation.     

A Redox-Dependent Mechanism for Regulation of AMPK Activation by Thioredoxin1 during Energy Starvation

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Nitrogen Starvation and the Regulation of Glutamine Synthetase in Agmenellum quadruplicatum

The level of glutamine synthetase activity in Agmenellum quadruplicatum strain PR-6 was dependent on the nitrogen source used for growth and on the nutritional status of the cells. During exponential growth, glutamine synthetase activity was low in cells grown on ammonia, urea, or nitrate. During the transition from nitrogen replete to nitrogen starved growth, glutamine synthetase activity began to rise. With ammonia as a nitrogen source, glutamine synthetase activity as determined in whole cells increased from 1 nanomole per minute per milliliter during exponential growth to 22 nanomoles per minute per milliliter during severe nitrogen starvation. In cells grown on nitrate the increase was from 5 to 39 nanomoles per minute per milliliter, and in cells grown on urea the increase was from 4 to 31 nanomoles per minute per milliliter.     

Regulation of Staphylococcal Enterotoxin B: Effect of Thiamine Starvation

During the transition between the exponential and stationary phases of growth, there was a rapid accumulation of both cell-associated and extracellular enterotoxin B. Extracellular enterotoxin was synthesized until the cells entered the stationary phase during which cell-bound toxin was not detected. The differential rate of toxin synthesis relative to that of total protein synthesis was greater at pH 7.7 than at 6.0. Addition of glucose decreased the differential rate of toxin synthesis. This decrease was greater at pH 7.7 than at 6.0. Addition of pyruvate decreased the differential rate at pH 7.7 but not at 6.0. Analysis of the nongaseous end products of glucose and pyruvate metabolism showed that conditions which favor the oxidative decarboxylation of pyruvate also favor the repression of toxin synthesis. Elimination of thiamine from the medium prevented the oxidative decarboxylation of pyruvate by Staphylococcus aureus S-6 and partially or completely reversed the repression of toxin synthesis by glucose and pyruvate. In the absence of an added energy source, thiamine starvation caused a decrease in protein synthesis but an increased differential rate of toxin synthesis which was greater at pH 7.7 than at 6.0. In the absence of thiamine, pyruvate was not metabolized but caused a decrease in the rate of protein synthesis. This resulted in a twofold increase in the differential rate of toxin synthesis. Thus, conditions which altered the oxidative decarboxylation of pyruvate or decreased the rate of protein synthesis increased the rate of enterotoxin B synthesis.