Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay an...Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay and Far Western Blotting assay, in vivo Co immunoprecipition assay were used. Results.The X199(51 99) domain of HBX is reponsible for HBX binding to TFIIB. While the d10 domain (125 295) of TFIIB is required for TFIIB binding to HBX. When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L), the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost. In vitro results of HBX binding to TFIIB were further confirmed by in vivo co immunoprecipitation assay. Our results also indicated that the Woodchuck hepatitis virus X protein (WHX) interacts with TFIIB. Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.展开更多
文摘Objective.In order to demonstrate the binding of HBV X protein (HBX) with the general transcription factor TFIIB. Methods.In vitro glutathion S transferase (GST) resin Pull Down assay and Far Western Blotting assay, in vivo Co immunoprecipition assay were used. Results.The X199(51 99) domain of HBX is reponsible for HBX binding to TFIIB. While the d10 domain (125 295) of TFIIB is required for TFIIB binding to HBX. When the two basic amino acids(K) at position 178 and 189 of TFIIB were substituted by neutral amino acids(L), the binding of TFIIBK178L and K189L to HBX was siginificantly reduced. When the the basic amino acids were substituted by the acidic amino acids(E),the binding of TFIIB K178E and K189E to HBX were almost lost. In vitro results of HBX binding to TFIIB were further confirmed by in vivo co immunoprecipitation assay. Our results also indicated that the Woodchuck hepatitis virus X protein (WHX) interacts with TFIIB. Conclusion.These results suggested that the communication between HBX and general transcription factor TFIIB is one of the mechanisms which account for its transcriptional transactivation.
