A new concept of the function of elongation factor 1 in peptid chain elongation.
(1/97)
An entirely new model for the mechanism of elongation factor 1 (EF-1) function is presented. Experiments in which mixtures of [3H]EF-1, ribosomes from Drebs II ascites cells and various additional co-factors were analyzed by chromatography on Sepharose 6B, show that EF-1 binds to the ribosome early in the translation process and remains bound on the ribosome during translation. Optimal EF-1 binding occurs on polynucleotide-programmed ribosomes carrying a tRNA in their P-site. On the other hand it was clearly shown that EF-2 attached at each translocation event and was then released before a new Phe-tRNA could be bound. (+info)
Control of the mRNA for hepatic tryptophan oxygenase during hormonal and substrate induction.
(2/97)
Glucocorticoid hormones increase the level of hepatic tryptophan oxygenase (EC 1.13.11.11; L-tryptophan:oxygen 2,3-oxidoreductase (decyclizing) by increasing its rate of synthesis. Studies were performed to determine whether this induction is mediated by controlling the level of the mRNA for tryptophan oxygenase of by changing the translational efficiency of a fixed level of mRNA. Activity of tryptophan oxygenase mRNA was quantitated in a Krebs ascites cell-free, protein-synthesizing system, supplemented with tRNA and rabbit reticulocyte initiation factors. De novo synthesis of the protomeric unit(s) of the enzyme was a linear function of the amount of mRNA added. Time course and dose-response studies in which the enzyme level and mRNA activity in livers from rats injected with inducing doses of hydrocortisone were compared indicate that the induction of this enzyme is accompanied by a proportional increase in the level of its mRNA. This was true for mRNA isolated from total liver as well as from cytoplasmic polysomes. Induction of the enzyme by its substrate, tryptophan, however, was not accompanied by a parallel increase in mRNA activity. (+info)
Isolation of rabbit reticulocyte initiation factors by means of heparin bound to sepharose.
(3/97)
Passage of cell-free extracts of rabbit reticulocytes through heparin-Sepharose affinity columns results in the loss of the ability of the effluent to initiate protein synthesis. This is shown by the loss of response to added rabbit globin mRNA or to inhibitors of initiation of protein synthesis, such as heparin and aurin tricarboxylic acid, and by recovery of initiation activity by addition of protein retained and subsequently eluted from the columns. The effluent retains, however, the ability to elongate protein chains. Only 0.8% of the applied cell extract protein binds to heparin-Sepharose columns. This bound protein, which can be recovered by increasing the salt concentration of the eluting buffer, has initiation factor activity equal to that of a crude initiation factor preparation obtained from rabbit reticulocyte ribosomes by extraction with 0.5 M KCl. The protein patterns on polyacrylamide gels of the initiation factors prepared by either method are very similar and indicate a protein mixture, which may represent a complex. These data confirm that heparin interacts specifically with initiation factos, and indicate that heparin-Sepharose chromatography will simplify procedures for the preparation of initiation factors. (+info)
Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.
(4/97)
An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains. (+info)
The binding of tritiated elongation factors 1 and 2 to ribosomes from Krebs II mouse ascites tumor cells.
(5/97)
Tritiated elongation factors 1 and 2 (EF-1 and EF-2) were obtained from Krebs II ascites cells which had been grown in mice injected with radioactive amino acids. The highly purified factors were sufficiently radioactive to be used in a study of the interactions between ribosomes and elongation factors. The following results were obtained. 1. EF-1 binding to ribosomes requires the presence of a polynucleotide, an aminoacyl-tRNA specified by the latter and a guanosine nucleotide carrying three phosphate groups. The hydrolysis of the GTP molecule involved in the binding reaction leads to the immediate release of EF-1. If GTP is replaced by Guo-5'-P2-CH2-P the factor remains bound to the ribosome and can be detected by sucrose gradient centrifugation techniques. 2. Likewise EF-2 binding to ribosomes can only be detected in the presence of GUO-5'-P2-CH2-P. 3. The affinity of ribosomes for EF-2 appears to be higher than for EF-1: PREINCUBATION OF RIBOSOMES WITH EF-2 inhibits the subsequent attachment of EF-1 almost completely. EF-1 prebound to ribosomes in the presence of GUO-5'-P2-CH2-P, POLY(URIDYLIC ACID) AND Phe-tRNA-Phe is partially removed from the ribosomes together with Phe-tRNA during a second incubation with EF-2. 4. Although EF-2 binding to ribosomes precludes any stable association between ribosomes and EF-1 it does not prevent the insertion of aminoacyl-tRNA into the ribosomal A-site. The attachment of aminoacyl-tRNA under these conditions enhances the binding of EF-2 to the ribosome. 5. The antibiotic showdomycin strongly inhibits the attachment of EF-1 to ribosomes and to a lesser degree impairs the binding of EF-2. 6. A-site ribosomes display a strong preference for the attachment of EF-2 and bind EF-1 only very poorly. The reverse is true for P-site ribosomes which are good substrates for the binding of EF-1 and bind EF-2 less efficiently than A-site ribosomes. These results and a number of additional findings made in this and in previous studies are discussed in the general context of the structure and function of mammalian elongation factors 1 and 2. (+info)
Isolation and characterization of collagen messenger RNA*.
(6/97)
Chick embryo collagen-synthesizing polysomes were isolated by differential centrifugation. RNA extracted from these particles was chromatographed in oligo(dT)-cellulose solumns and the mRNA thus obtained characterized as collagen mRNA by its electrophoetical mobility in acrylamide gels (equivalent to 1.05 x 10-6 daltons) and its effect upon a cell-free system derived from Krebs ascites tumor cells. The incorporation of 3H-proline was markedly dependent upon rabbit reticulocyte initiation factors and inhibited by initiation inhibitors such as aurintricaboxilate and pyrocatechol violet. The incorporation product was characterized as collagen by its lack of tryptophan, digestibility by purified bacterial collagenase, and by its co-chromatography with unlabled chick collagen in Sephadex G-200 and CM-cellulose columns. (+info)
Messenger activity of RNA transcribed in vitro by DNA-RNA polymerase associated to vaccinia virus cores.
(7/97)
The coding properties of RNA transcribed in vitro by purified vaccinia cores have been investigated using Krebs ascites tumor cells, L cells, and reticulocyte lysates. Six to 10 proteins synthesized in vitro are separated on polyacrylamide gels by electrophoresis in the presence of sodium dodecyl sulfate. Their molecular weights vary from 10,000 to 44,000. The electrophoretic behavior of these proteins is similar to that of early proteins isolated from infected L cells. The tryptic peptide analysis of one of these proteins indicates similarity in amino acid sequences. These results show fidelity of both in vitro transcription and molecular weight above 44,000 are synthesized in vitro does not seem due to a competition between 12S mRNA synthesized in excess and RNA of a higher sedimentation coefficient present in a lower amount. (+info)
Assay of protamine messenger RNA from rainbow trout testis.
(8/97)
A low molecular weight RNA fraction possessing protamine mRNA activity was prepared from rainbow trout testis polysomes. Addition of low molecular weight RNA to a Krebs II ascites S-30 cell-free protein synthesis system strongly stimulated [14C]arginine incorporation into acid-insoluble material. This stimulation was completely abolished by 10-4 M aurintricarboxylic acid, an inhibitor of eukaryotic protein synthesis at the level of initiation. Starch gel electrophoresis showed that labeled arginine was incorporated in vitro into products identical with both authentic protamine and histones as found previously (Gilmour, R. S., and Dixon, G. H. (1972) J. Biol. Chem. 247, 4621-4627). The 4 to 6 S RNA fraction, isolated from the polysomal low molecular weight RNA by sucrose gradient fractionation, enhanced the incorporation of [14C]arginine into acid-insoluble material and when this product was examined by starch gel electrophoresis, it co-migrated with authentic rainbow trout protamine. (+info)