| Summary: | 碩士 === 國立臺灣大學 === 生化學研究所 === 87 === The activity of thymidine kinase is increased in the S phase of the cell cycle. It is well documented that transcriptional and translational activation of cytosolic human thymidine kinase ( TK ) expression lead to the elevation of its activity at the G1-S transition. Here, we show that the 5'untranslated region ( 5'UTR ) of TK mRNA may have a functional role during its translation.
When the recombinant expression plasmid Flag-TK was transfected into 3T3 and Ltk-, both Flag-TK fusion protein and TK polypeptide were detected. A series of experiments were performed to indicate that the expression of TK from pFlag-TK is a result of alternative initiation. When the 5'UTR of TK gene was deleted from the pFlag-TK construct, alternative initiation of TK translation was reduced significantly. To investigate the possibility of the TK 5'UTR being an internal ribosomal entry site ( IRES ), we constructed a bicistronic expression plasmid Flag-GFP-TK for transfection experiment. We found that TK could no longer be expressed from this construct, indicating that the TK 5'UTR is not an IRES. We further examined the role of the TK 5'UTR in the in vitro translation system, and showed that the presence of the TK 5'UTR could cause the loss of cap dependency in translation. Treatment with rapamycin, which has been shown previously to inhibit cap-dependent translation, had no effect on the TK expression even though it reduced about 30﹪protein synthesis in IMR-90 cells during serum stimulation. Taken together, our results imply that the TK 5'UTR may serve as a regulatory cis-element for the translational control.
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