DNA-protein interactions between mammalian nuclear proteins and a GCC-element included in a composite cis-acting element of mouse ribosomal protein L32 promoter

DNA-protein complex formation between the sequence GC(GCC)(4) (GCC-element) of mouse ribosomal protein L32 (rpL32) promoter and nuclear proteins of mo use and human cells has been studied using gel retardation and South-Wester n blotting methods. The rpL32 promoter fragment (-24...+11) was able to for m specific complexes with mouse and human nuclear proteins mainly due to th e presence of the GCC-element (-19...-6). DNA-protein complex patterns exhi bited marked tissue-specificity. Three nuclear polypeptides of similar to 1 8, 28, and 50 kD that bind to the rpL32 promoter region (-24...+11) have be en detected in HeLa cells by ligand blotting. At least one of them (18 kD) interacted with the GCC-element directly. The same fragment of the promoter interacted only with one nuclear polypeptide (28-31 kD) from human fibrobl asts. DNA-protein complex formation between the investigated rpL32 promoter fragment containing the GCC-element and human fibroblast nuclear proteins is Zn2+-dependent. The method of functional titration (in vivo competition in the CAT-test) revealed that the GCC-element within the rpL32 promoter fu nctions as a positive cis-acting transcriptional element in NIH 3T3 cells. Thus, our data characterize the sequence GC(CCC)(4) as a functionally activ e cis-element included as a component in the more complex (composite) cis-e lement of mouse rpL32 promoter exhibiting tissue-specific properties. In va rious mammalian cell types the GCC-element can interact with various nuclea r proteins, and the mode of these interactions can be determined by its rel ative position to other cis-elements in the regulatory sites of the genome.