[In vitro modelling of the interactions between the promoter and enhancer complexes].
The aim of this work is to study in vitro mechanisms of interactions between the promoter and enhancer complexes of the transcriptional apparatus. We used retardation assay for the labeled TATA-box containing oligonucleotide with an unfractionated nuclear extract and with a second unlabeled oligonucleotide carrying an enhancer sequence. As an enhancer sequence we have chosen e5 element that is known to interact with members of Pax family transcription factors. We showed that the presence of unlabeled e5 element led to a significant decrease in the mobility of TATA-box associated complex. E5 element with a mutated homeodomen binding site does not produce such an effect, while a mutation in the paired binding site does not change the ability of e5 element to influence the formation of promoter-associated complex. We observed the same complex using crude nuclear extracts from cells that express chimeric protein Pax7-FKHR with higher molecular weight. The fact that the increase in the size of the transcription factor does not influence the size of the TATA-box associated complex led us to conclude that this complex did not result from association of original promoter and enhancer components, but rather the role of enhancer may be to stabilize the slowly migrating complex on the TATA-box via short-term interactions inducing conformational changes in the included proteins.
Kondrashov
A V
AV
Institute of Cytology RAS, 194064 St. Petersburg, Russia.
Pospelov
V A
VA
D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D051379:Mice; D009841:Oligonucleotides / Q000235:genetics; D051763:PAX7 Transcription Factor; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016385:TATA Box / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D018398:Homeodomain Proteins; D009841:Oligonucleotides; D051763:PAX7 Transcription Factor; C497321:PAX7 protein, human; C497322:Pax7 protein, mouse; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors
Modelirovanie in vitro transvzaimodeĭstviĭ mezhdu promotornym i énkhansernym kompleksami transkriptsionnogo apparata.