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 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 approximately 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.
Orlov
S V
SV
Institute of Experimental Medicine, Russian Academy of Medical Sciences, St. Petersburg, 197376, Russia.
Kuteikin
K B
KB
Dizhe
E B
EB
Kuryshev
V Y
VY
Shpakovich
V M
VM
Perevozchikov
A P
AP
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012269:Ribosomal Proteins; C041806:ribosomal protein L32; D004247:DNA