Demonstration of thyroxine-stimulated incorporation of amino acid into peptide linkage in mitochondria-free system.
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The observation that thyroxine stimulated in vitro protein synthesis in the absence of mitochondria (Carter, W.J., Faas, F.H., and Wynn, J (1971) J. Biol. Chem. 246, 4973-4977) has been disputed on the basis that radioactivity incorporated into protein did not represent peptide synthesis but incorporation of labeled contaminants present in the L-(U-14C) valine precursor (Sokoloff, L., and Roberts, P.A. (1972 Fed. Proc. 31, 1525). The question of mitochondrial requirement is important in determining whether thyroxine has a direct action on the polysome or causes the release of stimulatory factors from mitochondria. In this paper, thyroxine stimulation of peptide synthesis in mitochondria-free systems has been confirmed. Peptide synthesis is required for the thyroxine effect since it is dependent on the presence of polysomes and an energy source in the reaction mixture and is abolished by puromycin. The thyroxine effect is not due to incorporation of labeled contaminants since hydrolysis of labeled protein recovered from control and thyroxine-treated reaction mixtures yields the labeled amino acid precursor as the only radioactive product. Thyroxine stimulates polyuridylic acid-directed polyphenylalanine synthesis, providing further evidence that thyroxine is stimulating peptide synthesis rather than incorporation of radioactive contaminants by mechanisms other than peptide synthesis. Although thyroxine stimulates polyphenylalanine synthesis, it does not influence polyuridylic acid hydrolysis measured in the same reaction. Therefore, thyroxine stimulation of peptide synthesis is not due to prevention of hydrolysis of nucleic acid components of the reaction mixture. Thyroxine does not influence the size or specific activity of the free valine pool in the reaction mixture, indicating that observed increases in valine incorporation reflect increased peptide synthesis rather than increased specific activity of the valine precursor. The fact that thyroxine stimulates peptide synthesis using (14C)aminoacyl-tRNA precursors strengthens this conclusion. Therefore, thyroxine stimulation of protein labeling is dependent on the presence of peptide synthesis and cannot be explained by incorporation of labeled contaminants, prevention of RNA hydrolysis, or change in the specific activity of the amino acid precursor. Thyroxine causes a genuine increase in peptide synthesis by a direct action at the polysomal level. (+info)
Characterization of a 2',5'-oligoadenylate (2-5A)-dependent 37-kDa RNase L: azido photoaffinity labeling and 2-5A-dependent activation.
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Upregulation of key components of the 2',5'-oligoadenylate (2-5A) synthetase/RNase L pathway has been identified in extracts of peripheral blood mononuclear cells from individuals with chronic fatigue [corrected] syndrome, including the presence of a low molecular weight form of RNase L. In this study, analysis of 2',5'-Oligoadenylate (2-5A) binding and activation of the 80- and 37-kDa forms of RNase L has been completed utilizing photolabeling/immunoprecipitation and affinity assays, respectively. Saturation of photolabeling of the 80- and the 37-kDa RNase L with the 2-5A azido photoprobe, [(32)P]pApAp(8-azidoA), was achieved. Half-maximal photoinsertion of [(32)P]pApAp(8-azidoA) occurred at 3.7 x 10(-8) m for the 80-kDa RNase L and at 6.3 x 10(-8) m for the 37-kDa RNase L. Competition experiments using 100-fold excess unlabeled 2-5A photoaffinity probe, pApAp(8-azidoA), and authentic 2-5A (p(3)A(3)) resulted in complete protection against photolabeling, demonstrating that [(32)P]pApAp(8-azidoA) binds specifically to the 2-5A-binding site of the 80- and 37-kDa RNase L. The rate of RNA hydrolysis by the 37-kDa RNase L was three times faster than the 80-kDa RNase L. The data obtained from these 2-5A binding and 2-5A-dependent activation studies demonstrate the utility of [(32)P]pApAp(8-azidoA) for the detection of the 37-kDa RNase L in peripheral blood mononuclear cell extracts. (+info)
Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts.
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Messenger RNA (mRNA) of membrane-bound polysomes in a membrane fraction of WI-38 cells remains associated with the microsomal membranes even after ribosomes and their nascent polypeptide chains are removed by using puromycin in a high salt buffer or by disassembling the ribosomes in a medium of high ionic strength lacking magnesium. mRNA either was specifically labeled in the presence of actinomycin D, or it was recognized by virtue of its affinity for oligo-dT. Poly A segments in bound mRNAs have an electrophoretic mobility in acrylamide gels which is characteristic of cytoplasmic mRNAs and corresponds to 150-200 adenyl residues. Extensive RNase treatment did not lead to release of the poly A segments of membrane-associated mRNA molecules either from an intact membrane fraction or from a membrane fraction previously stripped of ribosomes. On the other hand, RNase treatment led to the release and digestion of the nonpoly A segments of the mRNA molecules, indicating that the site of attachment of mRNA to the ER membranes is located near or at the 3' end of the molecule which contains the poly A. A direct association of mRNAs and endoplasmic reticulum membranes is considered in a modelto explain the assembly of bound polysomes and protein synthesis in a membrane-associated apparatus. (+info)
Mutations in the G-domain of elongation factor G from Thermus thermophilus affect both its interaction with GTP and fusidic acid.
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Two hypersensitive and two resistant variants of elongation factor-G (EF-G) toward fusidic acid are studied in comparison with the wild type factor. All mutated proteins are active in a cell-free translation system and ribosome-dependent GTP hydrolysis. The EF-G variants with the Thr-84-->Ala or Asp-109-->Lys mutations bring about a strong resistance of EF-G to the antibiotic, whereas the EF-Gs with substitutions Gly-16-->Val or Glu-119-->Lys are the first examples of fusidic acid-hypersensitive factors. A correlation between fusidic acid resistance of EF-G mutants and their affinity to GTP are revealed in this study, although their interactions with GDP are not changed. Thus, fusidic acid-hypersensitive mutants have the high affinity to an uncleavable GTP analog, but the association of resistant mutants with GTP is decreased. The effects of either fusidic acid-sensitive or resistant mutations can be explained by the conformational changes in the EF-G molecule, which influence its GTP-binding center. The results presented in this paper indicate that fusidic acid-sensitive mutant factors have a conformation favorable for GTP binding and subsequent interaction with the ribosomes. (+info)
Identification of Sam68 arginine glycine-rich sequences capable of conferring nonspecific RNA binding to the GSG domain.
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Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. This family of proteins is often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. Here we performed a structure function analysis of Sam68 and identified two arginine glycine (RG)-rich regions that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, we demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids was sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, that minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. Our studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, our data lead us to propose that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated. (+info)
The RNA-binding specificity of the mouse Dazl protein.
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DAZ is an RNA-binding protein encoded by a region on the Y chromosome implicated in infertility, and DAZ-like (Dazl) proteins are master regulators of germ line gene expression in all animals. In mice Dazl is only expressed in germ cells and is necessary for meiosis. A dual approach was taken to understand the RNA-binding specificity of the Dazl protein: (i) traditional SELEX and (ii) a novel tri-hybrid screen. Both approaches led to the same conclusion, namely that Dazl binds oligo(U) stretches interspersed by G or C residues. In a directed tri-hybrid assay the strongest interaction was with the consensus (GUn)n. This motif is found in the 5' UTR of CDC25C whose homologue is thought to be the target of Boule, the Dazl homologue in flies. CDC25C 5' UTR also interacted specifically with Dazl in vitro. The tri-hybrid screen retrieved UTRs of known genes that may be physiological substrates of Dazl. (+info)
A mRNA determinant of gRNA-directed kinetoplastid editing.
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Several mitochondrial mRNAs of the kinetoplastid protozoa do not encode a functional open reading frame until they have been edited through the addition or deletion of U nucleotides at specific sites. Genetic information specifying the location and extent of editing is present on guide RNAs (gRNAs). The sequence adjacent to most mRNA editing sites has a high purine content which previously has been proposed to facilitate the editing reaction through base-pairing to a poly(U) tail at the 3' end of the gRNA. We demonstrate here that gRNA binding alone is insufficient to create an editing site and that the mRNA sequence near an editing site is an additional determinant affecting the efficiency of the reaction. (+info)
Isolation and partial characterization of a 40 S ribosomal subunit-transfer ribonucleic acid binding factor from rabbit reticulocytes.
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A factor that catalytically promotes the codon-directed, GTP-independent binding of tRNA to 40 S ribosomal subunits has been isolated from the postribosomal supernatant and the ribosomal wash of rabbit reticulocytes. The factor is a heat labile, sulfhydryl reagent-sensitive protein of a molecular weight of approximately 50,000. It consists of two non-identical subunits of Mr equals approximately 30,000 and 20,000. Its basic character has been confirmed by the high ratio of basic amino acids to nonamidic aspartic and glutamic acid present in the purified protein. Formation of a factor promoted 40 S-poly(U)-phenylalanyl-tRNA initiation complex causes a shift in the Mg-2+ concentration optimum for polyphenylalanine synthesis from 8 mM to 4mM. (+info)