Role in translation of a triple tandemly repeated sequence in the 5′-untranslated region of human thymidylate synthase mRNA

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Abstract
A triple tandem repeat (TTR) consisting of 90 nucleotides exists immediately upstream of the ATG initiator codon in human thymidylate synthase (TS) cDNA (pcHTS-1). To investigate the role of the TTR in the expression of the TS cDNA, we used pcHTS-1 to construct mutant cDNA clones in which part of the TTR was deleted or an additional element was inserted. The mutant cDNA plasmid was introduced into murine TS-negat1ve mutant cells and the relative translation efficiencies of the mutant cDNAs were determined by measuring the transient expression of TS activity and the amount of TS mRNA transcribed. The translation efficiency in transient expression of the mutants was increased by deletions covering all the first two repeated elements, and the part of the third closest to the ATG initiator codon, but was not affected by deletions of only parts of the first two repeated elements at the 5′ end. The translation efficiency was also not affected by insertion of an additional repeated element Into the TTR. These results suggest that the first two repeated elements at the 5′ end both have inhibitory effects on translation of the TS mRNA, probably due to the unique structural feature of this element.