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Abstract
A 67 kDa glycoprotein (p$\sp{67}$) protects the key peptide chain initiation factor eIF-2 $\alpha$-subunit from inhibitory phosphorylation in animal cells. This promotes protein synthesis even in presence of active eIF 2 kinases. In several cases studied, the p$\sp{67}$ level changes under different growth conditions in rat hepatoma (KRC-7) cells in culture. Results suggest that p$\sp{67 }$ is regulated at the mRNA level. The p$\sp{67}$ mRNA was present in confluent cells but disappeared almost completely from serum starved cells. However, when phorbol 13-myristate 12-acetate (PMA) was added to the same serum starved cells, p$\sp{67}$ mRNA appeared in increasing quantities. Further studies involving the expression of p$\sp{67}$ sense or antisense DNA in KRC-7 cells have provided evidence that the presence or absence of p$\sp{67}$ mRNA is the critical factor in regulating protein synthesis activity in these cells.
To gain a better understanding of the mechanism how p$\sp{67}$ gene is regulated, its promoter has been cloned and characterized. Sequence analysis of this 1.7 kilobase DNA fragment showed that the 898 base pairs at the 5$\sp\prime$-end of the promoter was identical to several LINE sequences. 148 base pairs at the 3$\sp\prime$-end contained the beginning of the first exon including the ATG initiator codon. The remaining 652 base pairs in between contained two AT-rich regions and several regulatory sequences. The mRNA initiation site was identified at 89 base pairs upstream from the ATG codon. Transcriptional regulation of the p$\sp{67}$ gene was also analyzed by transient transfection. When linked to a firefly luciferase reporter gene, this fragment enhanced transcription in KRC-7 cells. Using a series of deletions in the promoter, the minimum essential promoter region from $-$177 to $-$60 was identified. The promoter activity was also enhanced by treatment with PMA. This enhancement required an AP-1 sequence from $-$298 to $-$292 and a similar sequence form $-$97 to $-$88. Deletion of either of these sequences significantly reduced PMA enhancement. Deletion of both of these sequences almost completely eliminated PMA enhancement.
