Both TBP and TBP2 can promote transcription from TATA box containing promoters. (A) EMSA showing the binding of in vitro translated wild-type and abs-mutant TBP and TBP2 to a wild-type and a mutant TATA box probe. (B) A scheme of the experiment shown in (C) is presented. TBP-abs or TBP2-abs mRNA was injected into stage VI oocytes. After 4 hr oocytes were injected with either wild type or TGTA mutant hsp-70 promoter. pCMV-CAT was injected as an internal control. (C) Primer extension was performed to determine the expression from the hsp70 (hsp-70) and CMV promoter (CMV). Ctrl: RNA from un-injected oocytes. The bar graph shows the quantification of transcription signal from hsp-70 promoter normalized with that from CMV. The error bars represent standard error of mean from three independent experiments. (D) Western blot analysis of TBP2-wt, TBP-abs and TBP2-abs expression in oocytes for the experiment in (C). (E) Expression from the ZFP36L2 promoter with regular or mutant TATA box in the presence of TBP and TBP2 abs-mutants was analyzed by primer extension. The bar graph shows the quantification of transcription signal from ZFP36L2 promoter normalized with that from CMV from two independent experiments. (F) Western blot analysis of TBP-abs and TBP2-abs expression in oocytes for the experiment in (E). (G) Chromatin immunoprecipitation (ChIP) assay on germinal vesical-injected promoters from oocytes injected with HA-TBP or HA-TBP2 mRNA. Enrichment is the signal relative to the ChIP signal from oocytes not expressing HA-tagged protein.
Akhtar and Veenstra BMC Biology 2009 7:45 doi:10.1186/1741-7007-7-45