Roseann L. Shorey

Evidence for a guanine nucleotide-aminoacyl-RNA complex as an intermediate in the enzymatic transfer of aminoacyl-RNA to ribosomes

Abstract In recent investigations in this laboratory (1,2) two fractions required for polyuridylic acid-directed synthesis of polyphenylalanine were obtained from extracts of Escherichia coli W by chromatography on DEAE-Sephadex. One of the fractions, designated F-I, catalyzes the binding of aminoacyl-RNA to ribosomes in the presence of mRNA and GTP. This fraction has a high affinity for GTP and contains a GTPase that is stimulated by aminoacyl-RNA but not by stripped sRNA. Recently, Allende et al. (3) have shown that an enzyme fraction from E. coli B, which appears to be comparable to F-I, binds 3H-GTP. In the present investigation 3H-GTP was also found to interact with F-I to form a complex that is retained on a Millipore filter; however, if γ-32P-GTP is used, very little radioactivity is retained by the filter. On further investigation the amount of 3H-labeled compound bound to the protein was found to be decreased by the addition of aminocyl-RNA but not by stripped sRNA nor by N-acetylaminoacyl-RNA. Preliminary data indicate that F-I catalyzes the formation of a guanine nucleotide-aminoacyl-RNA complex which may be the intermediate product formed in the “enzymatic” transfer of aminoacyl-RNA to ribosomes.

A study of the enzymic transfer of aminoacyl-RNA to Escherichia coli ribosomes.

Abstract Three fractions, F-IA, F-IB, and F-II, have been obtained from extracts of Es- cherichia coli W by chromatography on DEAE-Sephadex. The guanosine 5′-triphosphate (GTP)-dependent transfer of aminoacyl-RNA to ribosomes is catalyzed by either F-IA or F-IB, and polypeptide synthesis is catalyzed by either fraction in the presence of F-II. Fraction F-IB is more labile than F-IA. Both F-IA and F-IB interact with GTP to form guanine nucleotide-enzyme complexes that are retained by Millipore filters. In the presence of aminoacyl-RNA, F-IA and F-IB interact with GTP to form a complex that is not retained by a Millipore filter. Guanosine 5′-diphosphate (GDP) also interacts with F-IA or F-IB to form a complex; however, no subsequent interaction with aminoacyl-RNA is observed. Both the GTP-dependent enzymic transfer of aminoacyl-RNA to ribosomes and the interaction of enzyme with GTP are inhibited by GDP. The enzymic transfer of aminoacyl-RNA to ribosomes is stimulated by NH 4 + and a sulfhydryl compound, and the interaction of the guanine nucleotide-enzyme complex with aminoacyl-RNA is also stimulated by NH 4 + . Deacylated sRNA, which inhibits nonenzymic binding, has no significant effect on the enzymic binding of aminoacyl-RNA to ribosomes. The guanine nucleotide-aminoacyl-RNA complex formed by the interaction of GTP, phenylalanyl-RNA, and either F-IA, F-IB, or a mixture of the two can be recovered by filtration through a Millipore filter and chromatography on Sephadex G-25. The active fractions from the Sephadex G-25 column contain close to stoichiometric amounts of phenylalanine and the guanine moiety and the -γ-phosphate moiety of the GTP. The amount of phenylalanine transferred to the ribosomes from the complex is 2-fold greater than the amount transferred from phenylalanyl-RNA alone and is equivalent to the amount transferred from phenylalanyl-RNA in the presence of enzyme and GTP. The guanine moiety of the complex is also transferred to the ribosomes, but no significant transfer of the γ-phosphate of the GTP present in the active fractions is observed.

[32] GTP-dependent binding of aminoacyl-tRNA to Escherichia coli Ribosomes

Publisher Summary This chapter describes the general procedures for preparation of ribosomes, [ 14 C]- or [3H]Phenylalanyl-tRNA, N-Acetyl-[ 14 C]phenylalanyl-tRNA, separation of TI s , TI u , and TII by DEAE-Sephadex chromatography, preparation of complex H, and binding of phenylalanyl-tRNA to ribosome.poly(U) complexes. Two transfer factors, TI s and Tl u , and GTP are required for the binding of aminoacyl-tRNA at the acceptor site of a ribosome-mRNA complex. Binding of [ 14 C]- or [ 3 H]aminoacyl-tRNA to the ribosomes is conveniently measured by adsorption of the ribosomal complex on a nitrocellulose (Millipore) filter. In the presence of aminoacyl-tRNA, TI s and TI u interact with GTP to form an aminoacyl-tRNA TI u GTP complex (complex II) that is not retained by a Millipore filter; TI s is still retained by the filter under these conditions. The binding of the phenylalanyl-tRNA moiety of complex II (prepared with [ 14 C]phenylalanyl-tRNA and a mixture of [ 3 H]GTP and [γ- 32 P]GTP) to a ribosome.poly (U) complex occurs at low concentrations of Mg 2+ and at low temperatures. When the phenylalanyl-tRNA moiety of complex II is bound at the acceptor site of a ribosome poly (U) complex carrying N-acetylphenylalanyl-tRNA at the donor site, peptide bond formation occurs.

The composition of the active intermediate in the transfer of aminoacyl-RNA to ribosomes

Abstract It was first shown in this laboratory (1,2) that one of the fractions obtained from extracts of Escherichia coli W by chromatography on DEAE-Sephadex catalyzes the GTP-dependent binding of aminoacyl-RNA to E. coli ribosomes in a manner analogous to that previously reported for the rabbit reticulocyte system (3,4). Further investigation (5,6) indicated that this fraction interacts with GTP in the presence of aminoacyl-RNA to form a complex which serves as the active intermediate in the enzymatic transfer of aminoacyl-RNA to E. coli ribosomes. Similar findings have also been reported by other investigators (7–12). Evidence has been obtained recently (13,14) that two protein fractions, differing in heat lability, are required for the formation of a GTP-protein complex and for the GTP-dependent transfer of aminoacyl-RNA to ribosomes. In the present investigation, evidence is presented which demonstrates that the active intermediate for the transfer of aminoacyl-RNA to ribosomes is a complex composed of aminoacyl-RNA, GTP, and the heat labile transfer factor.

Ethanol dependence produced in rats by nutritionally complete diets

Nutritionally complete diets formulated according to American Institute of Nutrition guidelines were used to make rats dependent upon ethanol. When intubated with a diet-ethanol solution for four days maintained initial body weight. When forced to consume the solution as the sole source of nutrients and water for nineteen days, rats gained weight. All animals developed severe withdrawal signs as measured by the intensity of tremors and spastic rigidity. The diet ingredients did not alter the absorption of the ethanol. The results demonstrate that physical dependence on ethanol can be induced in the rat without nutritional impairment.

The effect of guanylyl-5'-methylene diphosphonate on binding of aminoacyl-transfer ribonucleic acid to ribosomes.

The effect of guanylyl-5′-methylene diphosphonate (GMD) on the binding of Phe-tRNA to the acceptor site of a ribosome·poly (U)·AcPhe-tRNA complex and on the subsequent incorporation of the Phe-tRNA into peptide linkage has been studied. The binding that occurs in the presence of TIs, TIu, and GMD differs in several respects not only from that obtained in the presence of TIs, TIu, and GTP but also from that obtained in the absence of TIs and TIu, i.e., nonenzymatically. Nonenzymatic binding of Phe-tRNA to the acceptor site of the ribosome·poly (U)·AcPhe-tRNA complex occurs at high concentrations of Mg2+ (8–20 mM) and a significant portion of the Phe-tRNA interacts with AcPhe-tRNA to form AcPhe-Phe-tRNA. In the presence of TIs, TIu, and GTP, maximal binding of Phe-tRNA to the ribosomal complex occurs at low concentrations of Mg2+ (4–6 mM); the GTP is hydrolyzed; TIu·GDP, and Pi are released from the ribosome; and most of the Phe-tRNA bound to the ribosomes interacts with the AcPhe-tRNA to form dipeptide. In contrast, the concentration of Mg2+ required to obtain maximal binding of Phe-tRNA to the ribosomes in the presence of TIs, TIu, and GMD is about 12 mM; the TIu and GMD remain bound to the ribosomes; and very little dipeptide is formed. Moreover, the binding of Phe-tRNA to the ribosomes in the presence of GMD and transfer factors is very unstable. These data indicate that the presence of TIu and GMD on the ribosomes prevents the interaction of the Phe-tRNA at the acceptor site with the AcPhe-tRNA at the donor site, and suggest that hydrolysis of GTP and release of TIu and GDP from the ribosomes are prerequisites for peptide bond formation. In addition, evidence is presented which demonstrates that Phe-tRNA bound to the ribosomes in the presence of GMD is not incorporated into dipeptide upon the subsequent addition of GTP, indicating that the GMD bound to the ribosomes does not exchange with GTP.

The interaction of ethanol and swimming upon cardiac mass and mitochondrial function

Four groups of female Sprague-Dawley rats received a nutritionally adequate liquid diet formulated for rats. Two groups, one ethanol diet and one control diet swam 6 days/wk for 6 weeks and were designated swim ethanol (SWM-E) and swim control (SWM-C) respectively. Their swimming time increased from 15 min/day on the first day to 2 hrs/day during the final week. One sedentary group received an ethanol diet (SED-E) while another sedentary group received a control diet (SED-C). In the ethanol diet 35% of the calories as ethanol isoenergetically replaced dextrin. The group mean body weights were not different at the end of 6 weeks. The left ventricles of both swimming groups showed similar gains in weight, 13% for the ethanol and 15% for the control. Mitochondrial respiration in the ethanol groups showed a significant depression across substrates and across both pupulations of mitochondria (subsarcolemmal and intermyofibrillar). The swimming-ethanol interaction in the SWM-E group caused an atrophy of the gastrocnemius-plantaris muscle as evidenced by the 13% loss in weight of the muscle. We conclude that chronic ingestion of ethanol will suppress mitochondrial respiration in sedentary and swimming exercised rats, but will not suppress cardiac hypertrophy in the swimming exercised rats. Muscles that are not chronically overloaded by swimming, such as the gastrocnemius-plantaris muscles will undergo atrophy during the swimming protocol of 6 weeks.

Effects of different liquid diets and sustained ethanol release on alcohol metabolism

The effects of two liquid diets, Sustacal and Shorey-AIN, on liver alcohol dehydrogenase (ADH) activity and ethanol clearance were tested in rats under conditions of high ethanol exposure for nine days. High blood ethanol levels (BEL) were produced through a combination of an initial intubated dose of ethanol sustained ethanol release tube (SERT), and ethanol as 37% of total energy in the liquid diet. Under free-feeding conditions, rats consumed slightly more ethanol per unit body weight in the Shorey-AIN diet, a diet formulated for rodent nutrition, than in the Sustacal diet, a diet originally intended for human consumption. However, BEL were significantly higher in the Sustacal group than in the Shorey-AIN group. No differences in ethanol clearance rates were observed between the groups. On the other hand, total liver ADH activity was significantly reduced in both the Shorey AIN/ethanol and the Sustacal/ethanol groups, compared to lab chow controls. When the Sustacal diet was fortified with casein and methionine so that the protein content matched that of the Shorey AIN diet, the BEL were no longer significantly higher than those produced by the Shorey AIN/ethanol diet. The results demonstrate the effect of nutritional factors on BEL under conditions of high ethanol load. However, these factors do not appear to alter major characteristics of ethanol metabolism and clearance in our short-term experiments.