Richard Schweet

Isolation of a protein fraction from reticulocyte ribosomes required for de novo synthesis of hemoglobin

Abstract A fraction (I fraction) was prepared by extracting IX-reticulocyte ribosomes (unwashed) with 0.5 m KCl. This fraction is required for de novo synthesis of hemoglobin by various ribosomal preparations; however, it is not required for the completion of nascent chains. The active component(s) in the I fraction is protein in nature, since it is inactivated by pronase, trypsin, and N -ethylmaleimide (NEM) but is not affected by T 1 RNase. The active component(s) of the I fraction appears to have a molecular weight of at least 100,000. It appears to be involved in some step of protein synthesis following the formation of aminoacyl-tRNA but prior to the steps requiring the transfer enzymes (TF-1 and TF-2). The 1X-ribosomes are broken down into particles which sediment slower than 80S monosomes by the salt wash. In spite of this drastic treatment of 1X-ribosomes, the hemoglobin mRNA remains associated with the ribosomes.

ROLE OF THE GENETIC MESSAGE IN POLYRIBOSOME FUNCTION.

Studies of the properties of polyribosomes from puromycin-treated rabbit reticulocytes have supplied confirming evidence for the dynamic model of polyribosome function. A net, orderly increase of polyribosomes has been shown to occur in the cell-free system by attachment of monomeric ribosomes to the genetic message at the codon corresponding to the N-terminal amino acid of globin. Ribosomes which lose their growing peptide chain at any point along the messenger RNA can initiate a new chain at that point, and these incomplete chains can be released when the ribosome reaches the end of the genetic message. Thus we conclude that: (1) a polypeptide chain is normally initiated only at the N-terminal amino acid because an 80 s ribosome can attach only at the corresponding point on the messenger; (2) for release of a peptide chain it is both necessary and sufficient that the ribosome reach the end of the genetic message. The effect of puromycin on polyribosomes is not a direct one caused by the release of the growing peptide chain by puromycin, for the release can occur under conditions where the polyribosomal structures are unaffected by puromycin. While the synthesis of only a few peptide bonds will suffice for chain release by puromycin, much more extensive peptide bond formation must continue for puromycin to accelerate polyribosome breakdown. The action of puromycin is discussed in relation to modulation of the rate of protein synthesis, and it is proposed that the effect of puromycin on polyribosomes is due to an increased rate of travel of ribosomes along the genetic message.

STIMULATION OF AMINO ACID INCORPORATION IN AN ESCHERICHIA COLI CELL-FREE SYSTEM BY RETICULOCYTE RNA.

Abstract Stimulation of amino acid incorporation in an Escherichia coli cell-free system by added reticulocyte RNA has been studied. Soluble RNA was not effective, but ribosomal RNA gave an 8-fold stimulation of [ 14 C]valine incorporation. Attempts to isolate a specific messenger RNA fraction by washing reticulocyte ribosomes or fractionation of the isolated RNA were unsuccessful. The labeled soluble protein formed was characterized by column chromatography and the incorporation ratio of valine to isoleucine. It was concluded that only a small percentage, if any, of globin or large polypeptide precursors of globin was synthesized. The distribution of radio-activity in the labeled products was different, however, when reticulocyte and Escherichia coli ribosomal RNA were used to stimulate amino acid incorporation. The implications of these results were discussed.

Involvement of sulfhydryl groups in the binding of tRNA to reticulocyte ribosomes

Abstract The ability of purified reticulocyte ribosomes to bind phenylalanyl-RNA under the direction of polyuridylic acid is inhibited by a number of reagents reactive toward sulfhydryl groups; vicinal dithiols do not seem to be involved. This inhibition occurs in enzymic as well as non-enzymic binding and is also observed in the analogous lysyl-RNA polyadenylic acid system. N -Ethylmaleimide does not inactivate the bindin genzyme, nor does it react with ribosomal RNA. Instead, the site of inhibition is the ribosomal protein fraction. Also, sulfhydryl reagents do not seem to impair the ability of ribosomes to bind polyuridylic acid.

Studies on salt-treated reticulocyte ribosomes

Abstract Reticulocyte ribosomes were dissociated into metabolically active subunits by suspending the crude ribosomal preparation in 0.5 m KCl at a very high ribosome concentration (above 6 mg/ml). These subunits, which were harvested by sedimentation and stored in 0.25 m sucrose, are called shocked ribosomes (s-ribosomes). Structural transformations were exhibited when the ionic environment of the s-ribosomes was altered. In 0.1 m Tris-Cl, pH 7.5 (at 23 °) a suspension of s-ribosomes at a concentration of 4 mg/ml yields two major sedimenting species with sedimentation coefficients of 30S and 47S at 20 °. The relative concentration (by weight) of these subunits. was 1–2. The appearance of these two distinct subunits species depends on the relative concentration of Tris-Cl and ribosomes, and on the time of incubation. The degree of reconstitution into particles of 72S or larger parallels that of recovery of activity for amino acid incorporation.

Homopolynucleotide inhibition of poly U-dependent polyphenylalanine synthesis☆

Abstract Inhibition by homopolynucleotides of polyphenylalanine synthesis and phenylalanyl-RNA binding to ribosomes has been studied in a poly U-dependent, reticulocyte cell-free system. Inhibition by poly A was due to the formation of triple-stranded helical complexes with poly U, whereas poly I inhibited by attaching to ribosomes. Although poly A-poly U complex formation inhibited ribosome attachment to poly U, once attachment had occurred, the subsequent formation of A-U complexes did not inhibit peptide bond formation.

Purification of the transfer enzymes from reticulocytes and properties of the transfer reaction

Abstract The transfer of 14 C-amino acids from 14 C-aminoacyl-RNA into globin polypeptides on reticulocyte ribosomes required guanosine 5′-triphosphate (GTP) and two soluble enzyme fractions. The detailed purification of these transfer enzymes is described. The transfer reaction occurred at concentrations of MgCl 2 and KCl which were optimal for cell-free synthesis of hemoglobin. Other nucleoside triphosphates could not substitute for GTP. About 70% of the radioactivity of the ribosome-bound product was found in acid-precipitable polypeptides. The remaining 30% was found in an acid-soluble fraction containing mostly small peptides. A comparison suggests that amino acids are added on to existing polypeptide chains on the ribosomes in this hemoglobin transfer system, whereas chain initiation is the predominant reaction in a transfer system stimulated with polyuridylic acid.

Phenylalanyl puromycin formation as a model system for studying peptide bond formation

Abstract Using polyuridylic acid as a messenger RNA, phenylalanyl-RNA may be bound to two different sites on reticulocyte ribosomes. Phenylalanyl-RNA, bound to one of these sites, can react directly to form phenylalanyl puromycin. This reaction requires the peptide synthetase. Phenylalanyl-RNA in the other site requires GTP and possibly the binding enzyme as well as the peptide synthetase to participate in this peptide bond-forming reaction. The formation of phenylalanyl puromycin may be a convenient system to study various aspects of peptide bond formation. Various parameters of this model system are described. The rate-limiting step in the formation of phenylalanyl puromycin in this system is the binding of the peptide synthetase 3 to the ribosomes. Cycloheximide inhibits peptide bond formation by inhibiting association of the peptide synthetase with ribosomes.

Some properties of reticulocyte ribosomal subunits.

Abstract A method for preparing the subunits of reticulocyte ribosomes is described. These subunits are active for hemoglobin and polyphenylalanine synthesis. The subunits bind phenylalanyl-RNA enzymically using poly U as a messenger RNA. These three activities are a result of reassociation of the subunits into primarily monomeric ribosomes. The ability of the subunit preparation to participate in the above reactions is rapidly lost at 0 °. This loss of activity is paralleled by a loss in the ability to reassociate into monomeric ribosomes. The smaller 30S subunit can bind polyuridylic acid while nascent polyphenylalanine is bound to the 47S subunit.

Effect of varying the KCl and MgCl2 concentration on the enzymic and nonenzymic binding of phenylalanyl-RNA to reticulocyte ribosomes

Abstract Binding of phenylalanyl- 3 H-RNA to reticulocyte ribosomes in the presence of polyuridylic acid was studied using various concentrations of MgCl 2 and KCl. At salt conditions optimal for synthesis of polyphenylalanine (6.7 m m MgCl 2 and 67 m m KCl) a soluble fraction (binding enzyme) and guanosine 5′-triphosphate (GTP) were required for binding to occur. At higher concentrations of MgCl 2 or lower concentrations of KCl binding could occur without GTP and enzyme. Some of the phenylalanyl-RNA bound enzymically could be converted to diphenylalanine by incubation of the isolated ribosomes with a second enzyme (peptide synthetase). Phenylalanyl-RNA bound nonenzymically could be converted to the dipeptide only when GTP and the binding enzyme were included in this second incubation. A model of the ribosome with two binding sites for sRNA, the acceptor and donor sites, can be used to explain these results. Nonenzymic binding may occur predominantly, though not exclusively, in the acceptor site. Increasing the molar ratio of KCl to MgCl 2 could, as the result of changes in the ribosome shape, allow binding to occur readily in both sites; now GTP and the binding enzyme are required, perhaps to aid in the translocation of phenylalanyl-RNA from the acceptor to the donor site. A peptide bond may be formed between two phenylalanyl-RNAs in adjacent sites on the same ribosome. In the events which are prerequisite to formation of this first peptide bond, GTP and the binding enzyme appear to have another role in addition to aiding the physical binding of phenylalanyl-RNA to the ribosomes.