Protein Synthesis -Translation and Regulation
We showed recently that a mutant ofEscherichia coli initiator tRNA with a CAU--CUA anticodon sequence change can initiate protein synthesis from UAG by using formylglutamine instead of formylmethionine. We further showed that coupling of the anticodon sequence change to mutations in the acceptor stem that reduced Vma,jKmaPP in formylation of the tRNAs in vitro significantly reduced their activity in initiation in vivo. In this work, we have screened an E. coli genomic DNA library in a multicopy vector carrying one of the mutant tRNA genes and have found that the gene for E. coli methionyl-tRNA synthetase (MetRS) rescues, partially, the initiation defect of the mutant tRNA. For other mutant tRNAs, we have examined the effect of overproduction of MetRS on their activities in initiation and their aminoacylation and formylation in vivo. Some but not all of the tRNA mutants can be rescued. Those that cannot be rescued are extremely poor substrates for MetRS or the formylating enzyme. Overproduction ofMetRS also significantly increases the initiation activity of a tRNA mutant which can otherwise be aminoacylated with glutamine and fully formylated in vivo. We interpret these results as follows. (i) Mutant initiator tRNAs that are poor substrates for MetRS are aminoacylated in part with methionine when MetRS is overproduced. (ii) Mutant tRNAs aminoacylated with methionine are better substrates for the formylating enzyme in vivo than mutant tRNAs aminoacylated with glutamine. (iii) Mutant tRNAs carrying formylmethionine are significantly more active in initiation than those carrying formylglu-
nucleic acids & protein synthesis notes b1 - Biology …
Synthetic DL-methionine (DLM) supplements poultry diets to enhance production. The bioefficacy of liquid methionine is generally lower than that of powder methionine; however, if the level of total sulfur amino acids (TSAA) is set to the commercial recommendation, the bioefficacy of liquid methionine seems to be equal to that of powder methionine (equimolar basis). Absorption and transportation in the segment of the jejunum differ between liquid and powder methionine because multiple systems are involved. Methionine supplementation in a low-protein diet alleviates the negative effects of heat stress. The supplementation improves the amino acid balance and consequently promotes growth performance by enhancing feed efficiency, increases protein synthesis and decreases fat synthesis. Methionine supplementation also improves the immune response through direct effects (protein synthesis and breakdown) and indirect effects (derivatives of methionine). As various factors influence the methionine requirement, the requirements of commercial strains are higher than those recommended by NRC (1994). Moreover, the methionine requirement expressed as a percentage of diet declines during the starter and grower phases, while the requirement related to lysine is little changed (tends to increase).
Early passage human diploid fibroblasts develop senescent morphology prematurely within a week after a 2-hour pulse treatment with low or mild dose H2O2. We test here the role of cell cycle checkpoints, cytoskeletal proteins and de novo protein synthesis in senescent morphogenesis following H2O2 treatment. H2O2 treatment causes transient elevation of p53 protein and prolonged inhibition of Rb hyperphosphorylation. Expression of human papillomaviral E6 gene prevented elevation of p53 but did not affect senescent morphogenesis. Expression of human papillomaviral E7 gene reduced the level of Rb protein and prevented induction of senescent morphology by H2O2. The mutants of the E7 gene, in which the Rb family protein binding site was destroyed, could not reduce Rb protein or prevent H2O2 from inducing senescent morphology. Senescent-like cells showed enhanced actin stress fibers. In untreated cells, vinculin and paxillin preferentially distributed along the edge of the cells. In contrast, vinculin and paxillin distributed randomly and sporadically throughout senescent-like cells. E7 expression prevented enhancement of actin filament formation and redistribution of vinculin or paxillin. Neither wild-type nor E7 cells showed changes in the protein level of actin, vinculin or paxillin measured by western blot after H2O2 treatment. Finally, depletion of methionine in the culture medium after H2O2 treatment prevented senescent morphogenesis without affecting dephosphorylation of Rb protein. Our results suggest that senescent morphology likely develops by a program involving activated Rb family proteins, enhancement of actin stress fibers, redistribution of focal adhesion proteins and de novo protein synthesis.