U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Extraction of pure ribosomal protein and removal of Coomassie blue from acrylamide gels

A method has been developed to recover pure ribosomal proteins from two-dimensional polyacrylamide electrophoresis slabs. Proteins and Coomassie blue were extracted from the gels and separated by passing them through a Sephadex G-25 column. All the steps were performed in the presence of 66% ($\text{v}\text{v}$) acetic acid. The technique has been applied to Escherichia coll ribosomal proteins labeled with ¹²⁵I.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Variations on stoichiometry of ribosomal proteins in Escherichia coli

Experiments are described in which the Stoichiometry of the ribosomal proteins before and after ribosome release from mRNA is compared. Polysomes labeled with ³H (or ¹⁴C) and run-off 70 S particles (Subramanian el al., 1969) labeled with ¹⁴C (or ³H) were separately isolated, mixed, and the ribosomal proteins extracted and fractionated by two-dimensional gel electrophoresis. The measurement of the isotopic ratios shows that 47 proteins out of the 53 investigated are present in the same amount in polysomes as in run-off ribosomes indicating that they remain with the ribosome during the release step. Proteins S₁, S₂, S₆, S₂₁, $\text{L}{}_{\mathrm{<mtext>7</mtext>}}\text{L}{}_{\mathrm{<mtext>12</mtext>}}$ (Wittmann et al., 1971), however, show higher amounts in polysomes than in run-off ribosomes. The significance of these results is discussed.     

Effects of ethionine treatment on protein-synthesizing apparatus of rat liver 80 S ribosomes and 40 S ribosomal subunits

The inhibitory effects of ethionine treatment of female rats for 4 h on the protein-synthesizing machineries of 80 S ribosomes and 40 S ribosomal subunits of the liver were investigated. The following results were obtained. (1) The translation of globin mRNA by 80 S ribosomes or 40 S ribosomal subunits, in combination with mouse 60 S subunits, was markedly inhibited by ethionine treatment in a complete cell-free system containing partially purified initiation factors of rabbit reticulocytes and the rat liver pH 5 fraction. (2) The polysome formation of 80 S ribosomes in the complete system described above was inhibited by ethionine treatment. Similar inhibitions by ethionine treatment were observed in the case of incubation of 40 S subunits with reticulocyte lysate, although the polysome formation was rather low even in the case of control 40 S subunits. (3) The pattern of CsCl isopycnic centrifugation of rat liver native 40 S subunits uniformly labeled with [¹⁴C]- or [³H]orotic acid showed that the content of non-ribosomal proteins of native 40 S subunits was decreased by ethionine treatment. The analysis of proteins of native 40 S subunits by SDS-polyacrylamide slab gel electrophoresis revealed that eIF-3 subunits and two unidentified protein fractions of molecular weight of 2.3·10⁴ and 2.1·10⁴ were decreased in ethionine-treated rat liver. (4) 40 S subunits from ethionine-treated or control rat livers were labeled with $\text{N-[}{}^3\text{H]ethylmaleimide}$ or $\text{N-[}{}^{14}\text{C]ethylmaleimide}$, and the ³H to ¹⁴C ratios of individual 40 S proteins on two-dimensional polyacrylamide gel electrophoresis were measured. The results suggested that the conformation of rat liver 40 S subunits was changed by ethionine treatment. (5) These results may indicate that ethionine treatment decreases the activity of rat liver 40 S subunits for the interaction with initiation factors, especially eIF-3, as the results of conformational changes of 40 S subunits.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

The inhibition of eucaryotic protein synthesis by procaryotic elongation factor Tu

The activity of elongation factor Tu (EF-Tu) from $\text{Escherichia}\text{coli}$ in eucaryotic protein synthesis systems was investigated. EF-Tu was found to inhibit polyphenylalanine synthesis when incubated with $\text{Artemia}$ 80S ribosomes, purified rabbit reticulocyte elongation factor Tu (eEF-Tu) and partially purified reticulocyte translocase enzyme, eEF-G. The inhibition could be overcome by supplying the system with additional eEF-Tu. EF-Tu also inhibited protein synthesis in rabbit reticulocyte lysates. Data presented in this report indicate that inhibition by EF-Tu results from the accumulation of ternary complexes of the protein factor, GTP and aminoacyl-tRNA which do not interact with the ribosomal A-site of 80S ribosomes under physiological conditions.     

The mechanism of action of initiation factor 3 in protein synthesis. II. Association of the 30S ribosomal protein S12 with IF-3

Dimethylsuberimidate was used to crosslink ¹⁴C-labeled chain initiation factor 3 to $\text{E. coli}$ 30S particles. The crosslinked ribosomal proteins were analyzed by dodecyl sulfate polyacrylamide gel electrophoresis, and one major radioactive aggregate was found corresponding to a molecular weight of 41,000. Ribosomal protein S12 was identified to be crosslinked to IF-3 by immunological cross-reactivity.     

Mechanism of interferon action partial purification and characterization of a low-molecular-weight interferon-mediated inhibitor of translation with nucleolytic activity

A low-molecular-weight interferon-mediated ribosome-associated inhibitor of reovirus mRNA translation was purified from the 0.5 $\text{M}$ KCl ribosomal salt-wash fraction of mouse L₉₂₉ cells. The inhibitor possessed nucleolytic activity with reovirus [³H]mRNA as a substrate. Loss of translational inhibitory activity correlated with the thermal inactivation of the nuclease. A low-molecular-weight ($\text{<}$10K) component present in the Bio-Gel P150 chromatography fractions which contained the interferon-mediated nucleolytic activity was labeled in vivo with [¹⁴C]valine; a smaller component present in the same fractions was phosphorylated in vitro with [γ-³²P]ATP. The $\text{<}$10K components were resolved from ～50K, ～30K and ～20K phosphorylatable proteins associated with ribosomes that possess the interferon-mediated inhibitor(s) of viral mRNA translation.     

Incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into proteins and lipid intermediates in microsomal and golgi membranes from rat liver

Rough and smooth microsomes and Golgi membranes incorporate $\text{N-}\text{acetylglucosamine}$ from $\text{UDP}\text{-N-}\text{acetylglucosamine}$ into endogenous protein acceptors. A lipid intermediate of the dolichol phosphate type participates in this transfer reaction in the case of both microsomal subfractions, but the nature of lipid glycosylation is different in these two fractions. Glucosamine transfer in Golgi membranes does not appear to involve a lipid intermediate. In contrast to the results obtained under in vivo conditions, no glucosamine label is recovered in nascent ribosomal proteins or on luminal secretory proteins after incubation in vitro. Proteolysis of intact vesicles of the subfractions removes glycosylated dolichol phosphate and protein acceptors to various extents and interferes with transferase activities. This finding suggests the possibility that glycosylation at the cytoplasmic side of the membrane of the endoplasmic reticulum may involve a system separate from that acting at the luminal side of the same membrane.     

Identification of ubiquinone binding proteins in ubiquinol-cytochrome c reductase by arylazido ubiquinone derivative

A functionally active $\text{arylazido-1-[}{}^{14}\text{C]-β-alanine}$ ubiquinone derivative has been synthesized for the identification of the ubiquinone binding protein in ubiquinol-cytochrome $\text{c}$ reductase. After photolysis, the ¹⁴C activity was found to be specifically associated to proteins with mobilities relative to cytochrome $\text{c}$ of 0.841 and 0.475 in the sodium dodecylsulfate polyacrylamide gel electrophoresis of the Weber and Osborn system. These two proteins have previously been identified as $\text{b}$ cytochromes. The ¹⁴C activity distribution pattern was observed to be identical in the presence or absence of phospholipids during the photolysis. Antimycin A also produces no change in the ¹⁴C activity distribution among the proteins of this enzyme complex.     

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

One hour following administration of physiological concentrations of the steroid hormone antheridiol to a male strain of the water mold, $\text{Achlya}$$\text{ambisexualis}$, the rate of total cellular protein synthesis is increased. Further analysis revealed a sequential increase in the rate of syntheses for three classes of proteins following hormone stimulation. The rate of ribosomal protein synthesis increased as early as 20–30 minutes, followed by ribosomal salt wash proteins (40–60 minutes) and total soluble proteins after 60 minutes. Patterns of total cellular proteins, resolved by two-dimensional gel electrophoresis, during the first four hours after hormone treatment demonstrated the appearance of two newly synthesized peptides beginning at approximately 40 minutes followed by an increased rate of synthesis of three peptides after one hour. The synthesis of two peptides totally decreased after three hours of hormone induction.     

Crosslinking of proteins to ribosomal RNA in HeLa cell polysomes by sodium periodate.

HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in $\text{Escherichia}$,$\text{coli}$ 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs.     

Appearance of a kidney-specific ribosomal protein during mouse embryonic development

Ribosomes were prepared from adult mouse liver and kidney and the protein components examined by SDS polyacrylamide gel electrophoresis. A kidney specific protein was identified and was found to be associated with the large ribosomal subunit. $\text{In}$$\text{vitro}$ labelling of 11- and 14-day embryonic kidneys and subsequent analysis of the ribosomal proteins indicated a change in the ribosomal population during development. The kidney specific protein was synthesized during the first four days of kidney organogenesis.     

Ribosomal proteins L1, L17 and L27 from Escherichia coli localized at single sites on the large subunit by immune electron microscopy

Three-dimensional locations have been determined for Escherichia coli ribosomal proteins L1, L17 and L27 by immune electron microscopy using antibodies directed against these proteins. From the positions of immunoglobulin G attachment, observed in two characteristic projections, it was determined that these three proteins are located at single sites in different regions on the surface of the large subunit. In the quasisymmetric projection, L1 maps on the side opposite the “$\text{L}\text{7}\text{L}\text{12}$ stalk,” named the L1 ridge; protein L17 maps at the base of the subunit opposite the “central protuberance” (toward the $\text{L}\text{7}\text{L}\text{12}$ side of the subunit); and protein L27 is found on the central protuberance (on the side distal to the $\text{L}\text{7}\text{L}\text{12}$ stalk). In the asymmetric projection, proteins L1 and L27 are found on the surface of the subunit contracting the small subunit and protein L17 is on the surface of the subunit distal to the small subunit; i.e. on the cytoplasmic surface of the large subunit. Antibody binding at all three sites was eliminated when the immunoglobulin G molecules were preabsorbed with their specific proteins.     

Protein synthesis during a nutritional shift-up in Escherichia coli

The rate of synthesis of ribosomal protein increased immediately following a nutritional shift-up in $\text{Escherichia coli}$; while the rate of synthesis of elongation factors did not increase until 5–10 minutes had elapsed. The relative rates of synthesis of EFG and EFTs were constant at all times following shift-up. This constancy was not maintained between elongation factors and ribosomal protein early during shift-up. These data suggest that ribosomal and elongation factor proteins are not co-ordinately synthesized and argue against the postulate that their genes are part of one polycistron.     

Tubulin-like protein from Aspergillus nidulans

[³⁵S] labeled extracts of the fungus $\text{Aspergillus nidulans}$ were copolymerized with purified porcine brain tubulin. The [³⁵S] $\text{A. nidulans}$ protein which copurified with porcine microtubules was found to be similar to [³H] chick tubulin when the two were coelectrophoresed on several polyacrylamide gel electrophoresis systems. These results strongly suggest the presence in $\text{A. nidulans}$ of a tubulin-like protein.     

Separation of aminoacyl-transfer RNAs by polyacrylamide gel electrophoresis

A polyacrylamide gel electrophoresis system for separating $\text{E.}$$\text{coli}$ tRNAs and aminoacyl-tRNAs is described. The tRNA was separated into 6 discrete bands which contained varyin aamounts of tRNA and therefore varying numbers of tRNA species. In order to locate specific tRNAs, tRNA was charged with a ¹⁴C amino acid and the aminoacyl-tRNA was located by autoradiography. With several amino acids, 2 isoaccepting species were found. In total, 30 aminoacyl-tRNAs were located.     

Studies on compartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hapatocytes

The existence of metabolically distinct pools of $\text{S-}\text{adenosyl-L-methionine}$ in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-¹⁴C]methionine, a metabolically ‘stable’ pool was labeled. A subsequent short labeling with [methyl-³H]methionine selectively labeled a putative metabolically ‘labile’ pool. The existence of these distinguishable pools was ascertained by following the ³H and ¹⁴C label disappearance in $\text{S-}\text{adenosyl-L-methionine}$ during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of $\text{S-}\text{adenosyl-L-methionine}$. In both yeast and hepatocytes, the ${}^3\text{H}{}^{14}\text{C}$ ratio in $\text{S-}\text{adenosyl-L-methionine}$ decreased sharply. The individual ³H and ¹⁴C decrease in $\text{S-}\text{adenosyl-L-methionine}$ showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct $\text{S-}\text{adenosyl-L-methionine}$ pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’ pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’ pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The $\text{S-}\text{adenosyl-L-methionine}$ pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel ³H and ¹⁴C decreases in $\text{S-}\text{adenosyl-L-methionine}$.