Role of far upstream repressor elements controlling proto-Ha-ras gene transcription.

The far upstream region of the rat Ha-ras gene has been characterized to determine whether possible repressor sequences may control the low level of Ha-ras gene transcription from its TATA-less, GC-rich strong promoter. The chloramphenicol acetyl transferase (CAT) gene under the control of the 3.8-kb Ha-ras upstream promoter was minimally expressed in HeLa cells. Surprisingly, CAT gene expression was increased by the deletion of a 0.7-kb BglII fragment containing non-coding exon minus 2 and TATA box promoter elements located 1.7 kb upstream of the GC-rich strong promoter. Far upstream (CA)25 repeats also appeared to repress Ha-ras gene activity. Sequences within the 0.7-kb BglII fragment suppressed CAT gene expression when placed upstream of a heterologous thymidine kinase (tk) gene promoter. Repressor activity was further localized to a 160-bp AvrII-BglII sub-fragment. Gel shift assays identified two sequence-specific DNA binding proteins. The results demonstrated for the first time that far upstream repressor sequences control normal transcription of the Ha-ras proto-oncogene.