The occurrence of gamma-carboxyglutamic acid in mammalian ribosomes.

γ-carboxyglutamic acid (GLA) has been identified and estimated in mammalian ribosomes (mouse, rat, man, cultured cells) by amino acid analysis of total extracted protein. Assay results varied from 5.7–19.1 nMoles GLA/mg protein (6.0–9.9 residues GLA/1000 residues GLU), depending on purity and origin of ribosomes. GLA was not eliminated by purification. GLA in mouse hepatoma ribosomes was increased 3-fold upon preparation of puromycin-dissociated sub-units and sedimentation on gradients containing 1.0 M KCl. This tightly bound GLA (17.7 nMoles/mg sub-unit protein) could be present in one or more ribosomal proteins incorporated in the nucleoli, or in GLA-containing proteins acquired in the cytoplasm.     

Activity of Euglena gracilis chloroplast ribosomes with procaryotic and eucaryotic initiation factors

A method that permits the preparation of Euglena gracilis chloroplast 30 S ribosomal subunits that are largely free of endogenous initiation factors and that are active in the binding of fMet-tRNA in response to poly(A, U, G), has been developed. These 30 S subunits have been tested for activity in initiation complex formation with initiation factors from both procaryotes and eucaryotes. We have observed that Escherichia coli IF-2 binds fMet-tRNA nearly as well to Euglena chloroplast ribosomal subunits as it does to its homologous subunits. Neither wheat germ eIF-2 nor Euglena eIF-2A can bind fMet-tRNA efficiently to Euglena chloroplast or E. coli 30 S subunits although both are active with wheat germ 40 S ribosomal subunits. Euglena chloroplast 68 S ribosomes will also bind the initiator tRNA. Both E. coli IF-2 and E. coli IF-3 stimulate this reaction on chloroplast ribosomes with approximately the same efficiency as they do on their homologous ribosomes. E. coli IF-1 enhances the binding of fMet-tRNA to the chloroplast 68 S ribosomes when either IF-2 or IF-3 is limiting. The chloroplast ribosomes unlike E. coli ribosomes show considerable activity over a broad range of Mg2+ ion concentrations.     

Euglena gracilis chloroplast ribosomes: improved isolation procedure and comparison of elongation factor specificity with prokaryotic and eukaryotic ribosomes

An improved method for the isolation of Euglena chloroplast ribosomes is described which presents a number of advantages over past procedures. First, ribosomes are prepared from whole cell extracts, thus bypassing the need to isolate intact chloroplasts and resulting in a 10-fold improvement in yield. Second, the inclusion of 40 mm Mg²⁺ in the preparation buffers, while stabilizing the chloroplast ribosomes, precipitates and, thereby, virtually eliminates the cytoplasmic 89 S ribosomes. Third, greater than 95% of the chloroplast ribosomes sediment at 68 S rather than as the damaged 53 S particle frequently generated in other preparation procedures. Fourth, even with a high-salt wash to remove endogenous factors, the chloroplast ribosomes still sediment at 68 S and are just as active in in vitro protein synthesis as are E. coli ribosomes. These ribosomes have been tested for activity with elongation factors from prokaryotes, eukaryotes, and the chloroplast itself, and the results have been compared to those obtained with E. coli and wheat germ ribosomes. The data may be summarized as follows: (a) Chloroplast ribosomes use E. coli$\text{EF}\text{-}\text{Tu}\text{Ts}$ and EF-G with the same efficiency as do E. coli ribosomes in protein synthesis, (b) E. coli and chloroplast ribosomes can use Euglena chloroplast EF-G to catalyze translocation, but wheat germ ribosomes cannot, (c) Wheat germ EF-1_H and EF-2 are highly active in polymerization with wheat germ ribosomes, but ribosomes from neither E. coli nor the chloroplast are able to recognize these factors, (d) All three types of ribosomes accept Phe-tRNA from E. coli EF-Tu although to differing degrees. However, neither chloroplast nor E. coli ribosomes recognize wheat germ EF-1_H for the binding of Phe-tRNA.     

Translation of Synthetic and Endogenous Messenger Ribonucleic Acid In Vitro by Ribosomes and Polyribosomes from Clostridium pasteurianum

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.     

Purification and characterization of the mitochondrial translocase from Euglena gracilis

The Euglena gracilis mitochondrial protein biosynthetic elongation factor G (EF-Gmt) has been purified in four steps to greater than 50% homogeneity by use of a fusidic acid affinity procedure and conventional chromatographic techniques. The purification scheme results in 1100-fold purification with about 3% recovery of the total EF-G activity present in the postribosomal supernatant prepared from whole cell extracts. E. gracilis EF-Gmt has an approximate molecular weight of 76,000, comparable to that observed for procaryotic translocases. As is the case for other translocases which have been examined, pretreatment of E. gracilis EF-Gmt with N-ethylmaleimide results in a loss of polymerization activity, indicating a role for an essential cysteine residue in catalytic activity. GDP partially protects EF-Gmt from N-ethylmaleimide inactivation. E. gracilis EF-Gmt functions well on both Escherichia coli and E. gracilis chloroplast ribosomes, but has negligible activity on wheat germ cytoplasmic ribosomes. In this respect, it differs significantly from the mitochondrial translocase of yeast which has very little activity on chloroplast ribosomes. When assayed on E. coli ribosomes, E. gracilis EF-Gmt is sensitive to the steroid antibiotic, fusidic acid, at levels similar to that required for inactivation of E. coli EF-G. It is less sensitive than E. gracilis chloroplast EF-G, and is more sensitive than Bacillus subtilis EF-G. When assayed on E. gracilis chloroplast ribosomes, the same trends in sensitivities are observed, although the exact level of fusidic acid required for inactivation is slightly altered.     

Removal of wheat-germ agglutinin increases protein synthesis in wheat-germ extracts

Affinity chromatography of wheat germ extracts on a chitin column increased the rate and extent of protein synthesis, programmed by rabbit globin mRNA. Addition of purified wheat germ agglutinin to the chitin-treated extract reduced the rate of protein synthesis to about the levels seen in the untreated extracts. Experiments where the ratio of messenger to extract and the ratio of supernatant to ribosomes were varied, indicated that addition of wheat germ agglutinin reduced the amount of available ribosomes. Reduced and carboxymethylated wheat germ agglutinin failed to inhibit protein synthesis and was unable to bind to the ribosomes. However, labelled intact agglutinin was found to be bound to ribosomes. The bound agglutinin was not released by acid treatment. The inhibiting effect of wheat germ, agglutinin on protein synthesis could not be counteracted by addition of N-acetyl-D-glucosamine or sialic acid, whereas thiols partially diminished the inhibition. The data indicate that wheat germ agglutinin binds reversibly to ribosomes, probably through mixed disulfide formation, and that chitin treatment increases the ability of wheat germ extracts to support protein synthesis, at least in part, by removing the wheat germ agglutinin. The possibility that chitin treatment also removed other inhibitors of protein synthesis cannot be excluded.     

Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex

Animal mitochondrial protein synthesis factors elongation factor (EF) Tu and EF-Ts have been purified as an EF-Tu.Ts complex from crude extracts of bovine liver mitochondria. The mitochondrial complex has been purified 10,000-fold to near homogeneity by a combination of chromatographic procedures including high performance liquid chromatography. The mitochondrial EF-Tu.Ts complex is very stable and cannot be dissociated even in the presence of high concentrations of guanine nucleotides. No guanine nucleotide binding to this complex can be observed in the standard nitrocellulose filter binding assay. Mitochondrial EF-Ts activity can be detected by its ability to facilitate guanine nucleotide exchange with Escherichia coli EF-Tu. The EF-Tumt exhibits similar levels of activity on isolated mammalian mitochondrial and E. coli ribosomes, but displays minimal activity on Euglena gracilis chloroplast 70 S ribosomes and has no detectable activity on wheat germ cytoplasmic ribosomes. In contrast to the bacterial EF-Tu and the EF-Tu from the chloroplast of E. gracilis, the ability of the mitochondrial factor to catalyze polymerization is not inhibited by the antibiotic kirromycin.     

Purification and characterization of a ribosome dissociation factor (eukaryotic initiation factor 6) from wheat germ.

A wheat germ ribosome dissociation factor, eukaryotic initiation factor 6 (eIF-6), has been purified almost to homogeneity from the 25 to 40% ammonium sulfate fraction of the postribosomal supernatant. This dissociation factor is distinct from initiation factor eIF-3 and its chromatographic properties permit its separation from the known wheat germ initiation factors. Under certain conditions, eIF-6 stimulates the incorporation of amino acids into polypeptides in a partially fractionated wheat germ cell-free system. The eight-step purification procedure developed includes chromatography on DEAE-cellulose, phosphocellulose, Sephadex G-75, and hydroxyapatite and yields a dissociation factor more than 80% pure. The purified factor is composed of a single polypeptide chain with a molecular weight of approximately 23,000 as determined by gel filtration chromatography and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is an acidic protein which is heat labile and is inactivated by treatment with N-ethylmaleimide. The dissociation factor is much more effective in preventing the reassociation of 40 S and 60 S ribosomal subunits than in directly dissociating 80 S ribosomes. Like Escherichia coli IF-3, about 10 pmol of the dissociation factor are required to dissociate 1 pmol of ribosomes.     

Identification of cysteine-10 of protein S18 as part of the mRNA-binding site of Escherichia coli ribosomes by affinity-labeling studies with a chemically reactive A-U-G analog.

The reaction of a bromoacetamidophenyl derivative of the initiation codon A-U-G (A-U-G) with tight couples of Escherichia coli ribosomes leads to an exclusive crosslinking of label to protein S18. This crosslinking inhibits A-U-G-directed fMet-tRNAfMet binding into the puromycin-sensitive site of ribosomes and stimulates elongation-factor-dependent binding of Met-tRNAmMet. It is, therefore, concluded that protein S18 is located at or near the aminoacyl-tRNA binding site of E. coli ribosomes. Peptide as well as amino acid analysis shows that the reaction between A-U-G and ribosomes took place at cysteine-10 of protein S18. A-U-G could not be crosslinked to ribosomal proteins of the temperature-sensitive E. coli strain 258ts, where arginine-11 of protein S18 is replaced by a cysteine residue.     

Specific cleavage of ribosomal RNA caused by alpha sarcin.

Alpha sarcin causes the specific cleavage of RNA from 80S ribosomes and 60S subunits of yeast, but not from the 40S subunits to produce a small RNA fragment. The fragment was also produced on treatment of the 60S subunits of wheat germ ribosomes. The fragment has a molecular weight of 100,000 and is a cleavage product of the large RNA species in the 60S subunits. The fragment is not derived from the 5'end of the yeast 25S RNA nor does it bind to 5.8S RNA and we propose that the fragment represents the 3' terminal 320 nucleotides of 25S rRNA. The ability to produce fragment could not be separated from the ability of alpha sarcin to inhibit protein synthesis. Alpha sarcin also causes the specific cleavage of the 23S RNA of the E. coli subunit to produce a smaller fragment of RNA than that produced from eukaryote ribosomes.     

Translocation of proteins across the endoplasmic reticulum. I. Signal recognition protein (SRP) binds to in-vitro-assembled polysomes synthesizing secretory protein

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Wistar and Blobel 1980 Proc. Natl. Acad. Sci. U. S. A. 77:7112-7116). This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectively of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin); (b) to bind with relatively low affinity (apparent KD less than 5 x 10(-5)) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent KD less than 8 x 10(-9)) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low- and high- affinity binding as well as the selective translation-inhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High- affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue beta-hydroxy leucine was incorporated into the nascent secretory polypeptide.     

Overexpression of the methanococcal ribosomal protein L12 in Escherichia coli and its incorporation into halobacterial 50 S subunits yielding active ribosomes

The gene for the ribosomal L12 protein from the archaebacterium Methanococcus vannielii was cloned into the expression vector pKK223-3. The protein was overexpressed and remained stable in Escherichia coli XL1 cells. Purification yielded a protein with the same amino acid composition and sequence as in Methanococcus but it was acetylated at the N terminus as in the case with the homologous protein of E. coli. The in vivo incorporation of the overexpressed protein into the E. coli ribosomes was not observed. The overexpressed M. vannielii protein MvaL12e was incorporated into halobacterial ribosomes, thereby displacing the corresponding halobacterial L12 protein. Intact 70 S ribosomes were reconstituted from halobacterial 50 S subunits carrying the MvaL12e protein. These ribosomes were as active as native halobacterial ribosomes in a poly(U) assay. On the other hand, our attempts to incorporate L12 proteins from Bacillus stearothermophilus and E. coli into halobacterial ribosomes were not successful. These results support the conclusion which is based on primary sequence and predicted secondary structure comparisons that there exist two distinct L12 protein families, namely the eubacterial L12 protein family and the eukaryotic/archaebacterial L12 protein family.     

Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. I. Binding of tetracycline to components of the system. J. Bacteriol. 91:1917-1923. 1966.-Tetracycline, an inhibitor of cell-free protein synthesis, effected the dissociation of Escherichia coli 100S ribosomes to 70S particles in vivo and in vitro, but was not observed to mediate the further degradation of these particles. The antibiotic was bound by both 50S (Svedberg) and 30S subunits of 70S ribosomes and also by E. coli soluble RNA (sRNA), polyuridylic acid (poly U), and polyadenylic acid (poly A). The binding to ribosomal subunits was higher at 5 x 10(-4)m Mg(++) than at 10(-2)m Mg(++). The binding to polynucleotide chains was highest when Mg(++) was not added to the reaction mixture.     

Ribosomal proteins

Summary A comparison of the protein patterns of the 70S and 80S ribosomes from various plants, E. coli and yeast by disc-gel electrophoresis has shown the following relations: 1. There is a greater similarity between chloroplast ribosomes from various plants than between chloroplast and cytoplasmic ribosomes obtained from the same plant. 2. The protein patterns of the cytoplasmic ribosomes from bean, spinach and tobacco are more similar to each other than when compared to that of wheat germ. 3. At least one band is common to cytoplasmic ribosomes from all plants tested. 4. Only very few bands with identical mobilities are observed between chloroplast and E. coli ribosomes and between cytoplasmic plant and yeast ribosomes.     

Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli

The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by ubiquitin activating enzyme, E1. Previously, we purified and characterized multiple species of E1 from wheat germ. We now describe the isolation and characterization of a cDNA clone encoding E1 from wheat. This clone (UBA1) was isolated from a cDNA expression library with anti-wheat E1 antibodies. It contained an open reading frame coding for 1051 amino acids and directed the synthesis of a protein that comigrated with a wheat germ E1 of 117 kDa. UBA1 was confirmed as encoding E1 by (i) comparison of the peptide map of the protein product of UBA1 synthesized in Escherichia coli with that of purified E1 from wheat, and (ii) amino acid sequence identity of peptides generated from purified E1 with regions of the derived amino acid sequence of UBA1. The isolation of two additional cDNAs closely related to UBA1 indicated that E1 was encoded by a small gene family in wheat. Nonetheless, a single poly(A+) mRNA size class of 4 kilobases hybridized with UBA1. When expressed in E. coli, the product of UBA1 catalyzed the formation of a thiol ester linkage between ubiquitin and an ubiquitin carrier protein. The ability of E. coli containing UBA1 to synthesize an active protein will allow us to identify domains important for E1 function using in vitro mutagenesis.     

Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.     

Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system

Day, L. E. (Chas. Pfizer & Co., Inc., Groton, Conn.). Tetracycline inhibition of cell-free protein synthesis. II. Effect of the binding of tetracycline to the components of the system. J. Bacteriol. 92:197-203. 1966.-When tetracycline, an inhibitor of cell-free protein synthesis, was preincubated with each component of the Escherichia coli cell-free system, i.e., ribosomes, soluble ribonucleic acid (sRNA), polyuridylic acid (poly U), and S-100 (supernatant enzymes), only the ribosomal-bound antibiotic was inhibitory to the cell-free assay. Experiments designed to further localize the site of inhibition to either the 50S (Svedberg) or the 30S ribosomal subunit were not conclusive. Tritiated tetracycline (7-H(3)-tetracycline) was bound to isolated 50S ribosomes, and these were recombined with 30S subunits to form 70S ribosomes. When these ribosomes were dissociated and the subunits reisolated, the antibiotic was found with both the 50S and the 30S particles. The same results were observed when the tetracycline was initially bound to the 30S subunit.     

Some structural properties of 80S acid protein from pea ribosomes

The 80S acid protein from pea ribosomes similar to the L7/L12 protein from E. coli was studied. This protein was found to be rich in alanine (18 mol.%) and to contain an acid amino acids excess over basic ones, the ratio of basic amino acids to acid ones was 0.42. As in the case of other eukaryotic L7/L12 homologs studied, the N-terminal amino acid of the protein is methionine. Using the double immunodiffusion technique, no crossreaction of E. coli anti-L7/L12 with 80S acid protein from pea ribosomes was observed. It was assumed that the protein molecule contains conservative sites responsible for the specific functioning of eukaryotic L7/L12 homologs.     

Molecular and functional properties of protein SS1 from small ribosomal subunits of Streptomyces aureofaciens

Small ribosomal subunits of gram-positive cells of Streptomyces aureofaciens contain an acidic protein designated SS1. Purified protein SS1 has the same mobility in sodium dodecyl sulfate/polyacrylamide gel as ribosomal protein S1 of Escherichia coli (apparent Mr 68 000). Protein SS1 was dissected under mild conditions with trypsin and generated fragments were compared with well-characterized fragments of protein S1. The protein SS1 contains a structure homologous with the C-terminal fragment of protein S1. The affinity of protein SS1 to poly(U) is virtually identical with that of E. coli protein S1. In contrast to protein S1, the addition of SS1 to partially S1-depleted ribosomes of E. coli had no stimulatory effect on poly(U)-directed synthesis of polyphenylalanine. At molar excess of SS1 over ribosomes, the protein had comparable inhibitory effect on polypeptide synthesis as had S1 of E. coli. Ribosomes of S. aureofaciens required about one order of magnitude higher concentration of poly(U) for maximum synthetic activity than did ribosomes of E. coli. The addition of proteins SS1 or S1 to ribosomes of S. aureofaciens had no stimulatory effect on translation of poly(U). Our data indicate that the high-molecular-mass acidic protein SS1 of small ribosomal subunits of S. aureofaciens exhibits only a part of the functional properties of E. coli protein S1.