The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase.
A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39 degrees C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34 degrees C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39 degrees C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D007935:Leucine-tRNA Ligase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010053:Ovary; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000032:analysis; D011072:Poly U / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D013696:Temperature
D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D012333:RNA, Messenger; D011072:Poly U; D000604:Amino Acyl-tRNA Synthetases; D007935:Leucine-tRNA Ligase