Influence of HMG-1 and Adenovirus Oncoprotein E1A on Early Stages of Transcriptional Preinitiation Complex Assembly

Abstract

The TATA-binding protein (TBP) in the TFIID complex binds specifically to the TATA-box to initiate the stepwise assembly of the preinitiation complex (PIC) for RNA polymerase II transcription. Transcriptional activators and repressors compete with general transcription factors at each step to influence the course of the assembly. To investigate this process, the TBP⅐TATA complex was titrated with HMG-1 and the interaction monitored by electrophoretic mobility shift assays. The titration produced a ternary HMG-1⅐TBP⅐TATA complex, which exhibits increased mobility relative to the TBP⅐TATA complex. The addition of increasing levels of TFIIB to this complex results in the formation of the TFIIB⅐TBP⅐TATA complex. However, in the reverse titration, with very high mole ratios of HMG-1 present, TFIIB is not dissociated off and a complex is formed that contains all factors. The simultaneous addition of E1A to a mixture of TBP and TATA; or HMG-1, TBP, and TATA; or TFIIB, TBP, and TATA inhibits complex formation. On the other hand, E1A added to the pre-established complexes shows a significantly reduced capability to disrupt the complex. In add-back experiments with all complexes, increased levels of TBP re-established the complexes, indicating that the primary target for E1A in all complexes is TBP.

The Binding Interaction of HMG-1 with the TATA-binding Protein/TATA Complex

“…1B compares the binding of HMG-1 (lanes 3-7) and HMG-1(A-B) (lanes 9 -14) to the TBP/TATA complex. HMG-1 binding produces a complex with an increased mobility, as reported previously (24). The HMG-1/TBP/TATA complex is evident at an (HMG-1/TBP) molar ratio of 4, whereas it is the sole species at a molar ratio of 40.…”

“…This is due in large part to its high positive charge (ϩ22) relative to HMG-1, which has a more moderate negative charge (Ϫ8). These findings, coupled with those from previous studies (10,(12)(13)(14)(15)(16)(17)24), further highlight the distinctly different character of the domains in HMG-1, which may provide the basis for understanding its reported multifunctionalities in context-dependent transcription (40).…”

“…The Acidic C Terminus of HMG-1 Is Essential for Stable HMG-1/TBP/TATA Complex Formation-HMG-1 binds to TBP/TATA to form an EMSA-stable HMG-1/TBP/TATA complex, exhibiting a significantly greater stability than the TBP/ TATA complex (24). To investigate the extent to which the acidic C terminus of HMG-1 contributes to the stability of the complex, the binding of HMG-1-(1-215) to TBP was compared with that for the didomain HMG-1(A-B)-(1-176) that lacks the C terminus.…”

“…The EMSA procedures have been described (24) but involved reacting the DNA probe with the proteins of interest in binding buffer (24 mM Tris acetate, pH 8.0, 10% glycerol, 4 mM magnesium acetate, 50 mM potassium glutamate, 0.1 mM EDTA, 1 mM dithiothreitol, 0.01% Nonidet P-40, 4 mM spermidine, 5 g/ml poly(dG-dC), and 115 g/l bovine serum albumin) for 30 min at 30°C. All samples were electrophoresed in 0.35ϫ TBE buffer (TBE, Tris borate-EDTA buffer) at 200 V for ϳ1.5 h in 4-6% nondenaturing polyacrylamide gels at 4°C.…”

“…Isolation, Purification, and Characterization of Proteins-Calf thymus HMG-1 and HMG-2 proteins were purified in non-denaturing conditions using salt extraction, selective precipitation with ammonium sulfate, and further purification by high pressure liquid chromatography using a MonoQ column (23,24).…”