DNA--Protein Interactions between Mammalian Nuclear Proteins and a GCC-Element Included in a Composite cis-Acting Element of Mouse Ribosomal Protein L32 Promoter

S. V. Orlov, K. B. Kuteikin, E. B. Dizhe, V. Yu. Kuryshev, V. M. Shpakovich, and A. P. Perevozchikov^*

Institute of Experimental Medicine, Russian Academy of Medical Sciences, ul. Akademika Pavlova 12, St. Petersburg, 197376 Russia; fax: (812) 234-9489; E-mail: app@usr1.iem.ras.spb.ru

^* To whom correspondence should be addressed.

Received June 1, 1998; Revision received August 19, 1998

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DNA-protein complex formation between the sequence GC(GCC)₄ (GCC-element) of mouse ribosomal protein L32 (rpL32) promoter and nuclear proteins of mouse and human cells has been studied using gel retardation and South-Western blotting methods. The rpL32 promoter fragment (-24...+11) was able to form specific complexes with mouse and human nuclear proteins mainly due to the presence of the GCC-element (-19...-6). DNA-protein complex patterns exhibited marked tissue-specificity. Three nuclear polypeptides of ~18, 28, and 50 kD that bind to the rpL32 promoter region (-24...+11) have been 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 fibroblasts. 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 functions as a positive cis-acting transcriptional element in NIH 3T3 cells. Thus, our data characterize the sequence GC(GCC)₄ as a functionally active cis-element included as a component in the more complex (composite) cis-element of mouse rpL32 promoter exhibiting tissue-specific properties. In various mammalian cell types the GCC-element can interact with various nuclear proteins, and the mode of these interactions can be determined by its relative position to other cis-elements in the regulatory sites of the genome.

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KEY WORDS: mouse rpL32 gene, cis-elements, DNA-binding proteins, transcriptional factors, GCC-element

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