A structural and functional analysis of thec-ki-ras promoter and its binding proteins

The essential role of the ras genes in coupling receptors to intracellular signals required for cell growth and differentiation has been well established. Mutations in the coding region of the ras genes and the elevation of normal ras proteins have been found to cause tumor formation, indicating the quantitative and qualitative importance of the ras genes. The function of the ras proteins has been extensively studied. However, little is known about the regulation of ras gene expression. We used the mouse c-ki-ras gene as a model to investigate the transcriptional control of ras genes. Using a series of internal deletion mutants of the c-ki-ras promoter, two regulatory elements were identified. One has a negative effect while the other has a positive effect on c-ki-ras expression. The nucleotide substitution experiment and the structural analysis suggest that formation of the unusual triple-helix structure (H-DNA) in the homopurine-homopyrimidine region (CT-repeat) of the c-ki-ras promoter has a negative effect on its expression. Three c-ki-ras promoter binding proteins were isolated by southwestern screening. 101b binds the sequence which is identical between the mouse and the human c-ki-ras promoter. L1 binds the single-stranded region of H-DNA. KRZ, a zinc finger protein, binds the double-stranded CT-repeat. Together with the studies on c-myc gene expression, we proposed that the binding of 101b and KRZ activates while the binding of L1 inactivates c-ki-ras expression. Two forms of KRZ, KRZ1 and KRZ2, containing different numbers of zinc-finger motifs and the very C-terminal sequence were generated by alternative splicing. KRZ1 activates while KRZ2 represses the expression of c-ki-ras. Furthermore, KRZ1 is the predominant form in mouse tissue which is consistent with the fact that the tissues have more KRZ RNA containing more c-ki-ras transcripts. In summary, our results suggest that the promoter sequence of c-ki-ras, the secondary structure of this promoter, the interactions between promoter and its binding proteins, and the competition between the binding proteins are crucial for c-ki-ras expression