Functional analysis of TFIID-activator interaction by magnesium-agarose gel electrophoresis.
The general transcription factors TFIID and TFIIA are critical for the recognition of promoter start sites and mediate the stimulatory effect of some transcriptional activators. The regulation of TFIID binding to promoter DNA by activators and coactivators can be studied using a modified gel electrophoresis mobility shift assay (EMSA). TFIID is a multiprotein complex that consists of the TATA binding protein (TBP) and TBP associated factors (TAFs). TBP is a sequence-specific DNA binding protein that binds in the minor groove and introduces an energetically unfavorable bending angle of 100 degrees in the DNA. The activated preinitiation complex consists of TAFs, TBP, TFIIA, multiple activators, and approximately 200 bp of promoter DNA. The large mass and DNA distortions of the preinitiation complex preclude the use of conventional low ionic strength polyacrylamide gel EMSA for analysis. These large complexes can be analyzed by EMSA in agarose gels that contain magnesium ion. The Mg-agarose EMSA is a simple system useful for resolution of large multiprotein complexes that may introduce distortions in linear DNA. Important parameters are discussed so that this technique can be generally applied to other model activators.
Zerby
D
D
Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Lieberman
P M
PM
D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology; D004587:Electrophoresis, Agar Gel / Q000379:methods; D008274:Magnesium; D015341:Nucleic Acid Probes; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000502:physiology; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000502:physiology*; D015533:Transcriptional Activation; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology
C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D015341:Nucleic Acid Probes; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII; D014764:Viral Proteins; D008274:Magnesium