RESUMO
A genome-wide screen of a yeast non-essential gene-deletion library was used to identify sick phenotypes due to oxygen deprivation. The screen provided a manageable list of 384 potentially novel as well as known oxygen responding (anoxia-survival) genes. The gene-deletion mutants were further assayed for sensitivity to ferrozine and cobalt to obtain a subset of 34 oxygen-responsive candidate genes including the known hypoxic gene activator, MGA2. With each mutant in this subset a plasmid based ß-galactosidase assay was performed using the anoxic-inducible promoter from OLE1 gene, and 17 gene deletions were identified that inhibit induction under anaerobic conditions. Genetic interaction analysis for one of these mutants, the RNase-encoding POP2 gene, revealed synthetic sick interactions with a number of genes involved in oxygen sensing and response. Knockdown experiments for CNOT8, human homolog of POP2, reduced cell survival under low oxygen condition suggesting a similar function in human cells.
Assuntos
Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular/genética , Cobalto/farmacologia , Ácidos Graxos Dessaturases/genética , Ferrozina/farmacologia , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Humanos , Quelantes de Ferro/metabolismo , Proteínas de Membrana/genética , Oxigênio/metabolismo , Regiões Promotoras Genéticas , Ribonucleases/genética , Proteínas de Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase , Oligoelementos/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional , beta-Galactosidase/genéticaRESUMO
Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.