Exposure of the temperature-sensitive leucyl-tRNA synthetase mutant of Chinese hamster ovary cells, tsH to the non-permissive temperature of 39.5"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-2a subunit. It has previously been shown in other systems that phosphorylation of eIF-2a 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-2a by the reticulocyte haem-controlled repressor also inhibits eIF-2B activity in cell-free extracts derived from tsHl cells. Thus the observed increased phosphorylation of eIF-2a 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 fsH1 cells, TR-3 (which has normal leucyl-tRNA synthetase activity at 39.5"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 tRNALe" in vivo in tsHl cells at 39.5"C by treatment with a low concentration of cycloheximide failed to reverse the inhibition of eIF-2 function. This indicates that tRNA charging p e r 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 a 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 elF-2a, we have been unable to show any activation of kinase or inactivation of phosphatase following incubation of the cells at 39.5"C.Exposure of Chinese hamster ovary (CHO) cells containing temperature-sensitive aminoacyl-tRNA synthetase mutations to non-permissive conditions produces a rapid inhibition of the initiation of protein synthesis [l]. Using a temperature-sensitive leucyl-tRNA synthetase mutant, t s H l , we have shown that this is due to inhibition of the ability of initiation factor eIF-2 to form eIF-2 . GTP . Met-tRNA, ternary complexes and 43s pre-initiation complexes on native small ribosomal subunits [2]. Such impairment of eIF-2 activity is a consequence of a reduction in the rate of regeneration of the binary complex, eIF-2 . GTP, from eIF-2 . GDP by the guanine nucleotide exchange factor, eIF-2B (sometimes known as GEF). This reduces the availability of eIF-2 . GTP to participate in ternary complex formation with the initiator Met-tRNAY" and consequently in the formation of the 43s ribosomal complexes necessary to initiate mRNA binding [3]. The inh...

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.

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This work was supported by a grant from the Medical Research Council. We thank Drs. H. B. F. Dixon and J. Siekierka for the gift of materials used in these experiments.