Audrey Stevens

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes

Hal2p is an enzyme that converts pAp (adenosine 3′,5′ bisphosphate), a product of sulfate assimilation, into 5′ AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5′→3′ exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild‐type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5′ processing of the 5.8S rRNA and snoRNAs, degradation of pre‐rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

Beta -Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

Previous work showed that human β-globin mRNAs harboring a premature termination codon are degraded in the erythroid tissues of mice to products that lack sequences from the mRNA 5′ end but contain a 5′ cap-like structure. Whether these decay products are the consequence of endonucleolytic or 5′-to-3′ exonucleolytic activity is unclear. We report that this β-globin mRNA decay pathway is recapitulated in cultured mouse erythroleukemia (MEL) cells and targets nonsense-free mRNA to a lesser extent than nonsense-containing mRNA. S1 nuclease mapping and primer extension demonstrated that 70–80% of decay product 5′ ends contain a UG dinucleotide. Detection of upstream counterparts of these decay products indicates that they are generated by endonucleolytic activity. Both crude and partially purified polysome extracts prepared from MEL cells contain an endonucleolytic activity that generates decay products comparable to those observed in vivo. These data suggest that an endonuclease with preference for UG dinucleotides is involved in the degradation of nonsense-containing and, to a lesser extent, nonsense-free human β-globin mRNAs in mouse erythroid cells.

Analogs of human epidermal growth factor which partially inhibit the growth factor-dependent protein-tyrosine kinase activity of the epidermal growth factor receptor

Three site‐directed mutants of human epidermal growth factor, Leu‐26 → Gly, Leu‐47 → Ala, and He‐23 → Thr, were examined for their ability to stimulate the protein‐tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding affinities of the mutant growth factors were 20‐ to 50‐fold lower, as compared to wild‐type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial ‘uncoupling’ of signal transduction. The mutant analogs were shown to compete directly with the binding of wild‐type, resulting in a decrease in growth factor‐stimulated kinase activity.

Inhibition of DNA-enzyme binding by an RNA polymerase inhibitor from T4 phage-infected Escherichia coli.

Two different methods have been used to study the effect of an RNA polymerase inhibitor on DNA-enzyme binding. They show that the inhibitor, isolated from T4 phage-infected Escherichia coli, prevents binding of RNA polymerase to T4 and T7 DNA.

5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole inhibits a HeLa protein kinase that phosphorylates an RNA polymerase II-derived peptide☆

A protein kinase that phosphorylates Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4, a synthetic peptide homologous to the evolutionarily-conserved, tandemly-repeated heptapeptide sequence at the C-terminus of the large subunit of eukaryotic RNA polymerase II, has been detected in HeLa cell extracts and chromatographic fractions therefrom. The enzyme, which phosphorylates serine principally, can be distinguished from previously described major protein kinases which phosphorylate the peptide poorly, if at all. It is inhibited by the nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Results suggest that human placental RNA polymerase II is phosphorylated at the C-terminus of the large subunit by the partially-purified protein kinase and that the phosphorylation is also sensitive to the nucleoside analog.

An inhibitor of host sigma-stimulated core enzyme activity that purifies with DNA-dependent RNA polymerase of E. coli following T4 phage infection.

Abstract The content of the sigma subunit (as detected by gel electrophoresis) and activity with T4 DNA were examined with RNA polymerase fractions from both normal and T4 phage-infected E. coli . Sigma-containing fractions and core enzymes were obtained by phosphocellulose column chromatography. The sigma-containing fraction of the enzyme from infected cells, although somewhat stimulatory to both core enzymes alone, inhibits the normal sigma-stimulated activity of the core enzyme from infected cells at both low and high KCl concentration. Normal core enzyme activity is inhibited only at high KCl concentration.

An mRNA decapping enzyme from ribosomes of Saccharomyces cerevisiae.

Abstract By use of [ 3 H]methyl-5′-capped [ 14 C]mRNA from yeast as a substrate, a decapping enzyme activity has been detected in enzyme fractions derived from a high salt wash of ribosomes of Saccharomyces cerevisiae . The product of the decapping reaction is [ 3 H]m 7 GDP. That the enzyme is not a non-specific pyrophosphatase is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed.

An isotopic study of DNA-dependent RNA polymerase of E. Coli following T4 phage infection☆

Abstract When either 14 C-glucose or a 14 C-amino acid hydrolysate is added to a 3 H-amino acid-labeled culture of E. coli B one minute after T4am82 phage infection, both 3 H- and 14 C-label are found in highly purified DNA-dependent RNA polymerase. Following phosphocellulose column chromatography, more than 95% of the 14 C-label associated with the enzyme is found in a component with a molecular weight of about 10, 000, as analyzed by Sephadex G-200 column chromatography in the presence of sodium dodecyl sulfate. Under the conditions of these experiments, the phage infection caused no preferential loss of 3 H-label from the α as compared to the β subunit, and no significant incorporation of 14 C into either of them.

Inhibition of DNA-dependent rna polymerase of E.coli by phospholipids*

Summary DNA-dependent RNA polymerase of E. coli is strongly inhibited by phosphatidylglycerol and cardiolipin at concentrations of 5 to 8 × 10−5 M. The two lipids inhibit the formation of rifampicin-resistant preinitiation complexes. Phosphatidylethanolamine is not inhibitory, and at a tenfold higher concentration partially overcomes the inhibition by the other two lipids.

An analysis in vivo of histidine transfer RNA during repression and derepression in Bacillus subtilis

Abstract Bacillus subtilis 30 contains two iso-accepting species of tRNAHis that are relatively labile to hydrolysis when aminoacylated. The two species, when studied under repressive and derepressive states of the histidine biosynthetic enzymes, are qualitatively and quantitatively equivalent. However, derepressed cells have an average of 66 % of the histidyl-tRNA present in repressed cells in vivo. This difference is not due to the total amount of tRNAHis present in the two states but may reflect differences in the aminoacylation reaction in vivo.