AP-4- and AP-5-like proteins from mouse L cells are trans-activators and bind to the GT-II region of SV40 early TRE in a mutually exclusive manner.
Plasmids containing the cat reporter gene, transcription of which is directed by deletion mutants of the SV40 early region transcriptional regulatory element (SV40E TRE), were transfected into mouse L cells to determine the DNA motifs of SV40E TRE responsible for maximal gene expression. One deletion mutant, pSVE305, demonstrated a 50% reduction in CAT activity as compared to pSVE338, suggesting the importance of these 33 bp in directing efficient gene expression in mouse L cells. Introduction of triplet point mutations in this region and subsequent transfection studies in mouse L cells revealed three sites which were responsible for the reduction of CAT activity. These three mutations were located in the middle of the binding sites of three trans-activators: AP-3, AP-4 and AP-5. While the levels of CAT activity directed by SV40E TRE deletion mutants were similar in both HeLa and mouse L cells, the profiles of point mutants were different, suggesting that the activating ability of each nuclear factor is different from that of its counterpart in these two cell lines. Electrophoretic mobility shift assays (EMSA) demonstrated that binding of AP-4- and AP-5-like proteins of mouse L and HeLa cells to the GT-II motif occurs in a mutually exclusive manner. Furthermore, we observed a 'reverse competition' binding phenomenon which suggested a unique relationship between AP-4- and AP-5-like proteins of mouse L cells to the GT-II motif. Proteolytic mobility-shift analyses showed that an AP-5-like protein was more resistant to proteolytic digestion than an AP-4-like protein of mouse L cells.
Hou
Y T
YT
Department of Biological Sciences, Ohio University, Athens 45701, USA.
Coleman
T A
TA
Kopchick
J J
JJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015967:Gene Expression Regulation, Viral; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D015534:Trans-Activators; D014157:Transcription Factors; C055473:enhancer-binding protein AP-4; C095901:transcription factor AP-5