A novel role for aminoacyl‐tRNA synthetases in the regulation of polypeptide chain initiation

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Abstract
Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH1, to the non-permissive temperature of 39.5.degree.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 .cntdot. GTP .cntdot. 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 .cntdot. GDP to eIF-2 .cntdot. 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.degree.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.degree.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.degree.C.