Localization of a cycloheximide-sensitivity response element in the human thymidine kinase gene promoter

In order to localize the segment of the human thymidine kinase (TK) gene promoter that mediates sensitivity of TK mRNA expression to the presence of cycloheximide (CX), a series of promoter truncation mutants was prepared between the 460-base pair (bp) promoter that was demonstrated previously to be sensitive to CX and the 83-bp promoter that was demonstrated previously to be insensitive to CX. TK promoters containing 370, 300, 160, or 130 bp of 5'-flanking sequence were all sensitive to inhibition by CX. Further truncation to 100 bp of 5'-flanking sequence eliminated CX sensitivity. Electrophoretic mobility shift assays using a probe containing most of this region (but omitting the SP1 binding site at the 5'-end of the 130-bp promoter) identified some complexes whose formation was sensitive to the presence of CX. Comparison of the sequences of oligonucleotides that were able to compete for formation of mobility shift complexes identified the sequence GCGGCC as a putative CX-sensitivity response element. Two such sequences are found between 83 and 130 bp 5' of the TK capsite. Mutation of the distal sequence attenuated sensitivity of TK mRNA expression to CX, while mutation of the proximal sequence had minimal effect on CX sensitivity. Thus, these data have localized a CX-sensitivity response element to a segment of TK promoter about 120 bp 5' of the capsite that includes the hexamer GCGGCC.