title: A novel role for aminoacyl-tRNA synthetases in the regulation of polypeptide chain initiation.
abstract: Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5 degrees C results in a rapid inhibition of polypeptide chain initiation. This inhibition is caused by a reduced ability of the eukaryotic initiation factor eIF-2 to participate in the formation of eIF-2.GTP.Met-tRNAf ternary complexes and thus in the formation of 43S ribosomal pre-initiation complexes. Associated with this decreased eIF-2 activity is an increased phosphorylation of the eIF-2 alpha subunit. It has previously been shown in other systems that phosphorylation of eIF-2 alpha slows the rate of recycling of eIF-2.GDP to eIF-2.GTP catalysed by the guanine nucleotide exchange factor eIF-2B. We show here that phosphorylation of eIF-2 alpha by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsH1 cells. Thus the observed increased phosphorylation of eIF-2 alpha at the non-permissive temperature in this system is consistent with impaired recycling of eIF-2 in vivo. Using a single-step temperature revertant of tsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5 degrees C), we demonstrate here that all inhibition of eIF-2 function reverts together with the synthetase mutation. This establishes the close link between synthetase function and eIF-2 activity. In contrast, recharging tRNALeu in vivo in tsH1 cells at 39.5 degrees C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging per se is not involved in the regulatory mechanism. Our data indicate a novel role for aminoacyl-tRNA synthetases in the regulation of eIF-2 function mediated through phosphorylation of the alpha subunit of this factor. However, in spite of the fact that cell-free extracts from Chinese hamster ovary cells contain protein kinase and phosphatase activities active against either exogenous or endogenous eIF-2 alpha, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5 degrees C.
authors.affiliation: Department of Biochemistry, King's College, London.
authors.forename: J W
authors.initials: JW
authors.lastname: Pollard
authors.forename: A R
authors.initials: AR
authors.lastname: Galpine
authors.forename: M J
authors.initials: MJ
authors.lastname: Clemens
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2; D009154:Mutation; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D012356:RNA, Transfer, Leu / Q000378:metabolism; D013696:Temperature; D019892:eIF-2 Kinase
chemical_list: D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D012356:RNA, Transfer, Leu; D003513:Cycloheximide; D011494:Protein Kinases; D019892:eIF-2 Kinase; D010744:Phosphoric Monoester Hydrolases; D000604:Amino Acyl-tRNA Synthetases