The −83G>C mutation in the PK-R promoter strongly down-regulates promoter activity in vitro.

Luciferase reporter gene constructs containing 469 bp of the wild-type or mutated PK-R promoter were transiently transfected in K562 erythroleukemic cells. Luciferase activities were expressed relative to control pGL3-SV40 and pGL3-Basic was included as a negative (promoterless) control. (A) Constructs pGL3_PKRWT and pGL3_PKR248delT contained the wild-type or polymorphic −248delT allele, respectively. The latter mutation disrupts an inverted GATA-1 binding site (arrow) but no down-regulation of promoter strength is observed. In contrast, an increase in promoter activity was observed upon removal of thymine −248. (B) Individual and combined effects of the −83G>C and −324T>A missense mutations were studied using constructs that contained only the −83G>C mutation (pGL3_PKR83C) or the −324T>A mutation (pGL3_PKR324A), or both mutations in cis (pGL3_PKR324A/83C). The −324T>A mutation had no effect on promoter activity as compared with the wild-type (pGL3_PKRWT). In contrast, the −83G>C mutation is capable of strongly reducing in vitro promoter activity and is unaffected by the concomitant presence of the −324T>A substitution in cis. * Statistically significant (P < .05).