Molecular aspects of estrogen receptor activation factor (E-RAF) function.

Abstract

The estrogen receptor activation factor (E-RAF)-mediated binding of the receptor-estrogen complex to uterine nuclei was found to involve at least two classes of nuclear macromolecules: (1) the DNA, and (2) a proteinacious component. Evidences are presented to show that at least a portion of (2) is represented by the nuclear RNA polymerases. The receptor-estrogen complex associated in vivo with the nuclear RNA polymerases existed in two distinct forms which sedimented at 3.8 S and 4.8 S on sucrose density gradients. Almost 2/3 of the total radioactivity was associated with the 3.8 S species. Saturation kinetics of the two forms showed that while the 4.8 S form displayed characteristics similar to the classical type I nuclear binding site, the features displayed by the 3.8 S form were closely similar to those of the nuclear type II site. The 4.8 S species is a DNA binding form while the 3.8 S form is non-DNA binding. Anti-E-RAF IIA IgG cross-reacted with both the binding components. Goat uterine E-RAF I, IIA and IIB were purified to homogeneity as described earlier. While E-RAF IIA and IIB destabilized the native DNA structure and induced separation of the DNA strands, E-RAF I performed the opposite function. The reactions required the presence of ATP; all three of them displayed DNA-dependent ATPase activity. In an in vitro transcription system which contained purified RNA polymerase B (rat liver enzyme) and goat uterine DNA, E-RAF IIA and IIB enhanced transcription 7-fold over the control while E-RAF I totally suppressed the transcription process, irrespective of whether it was stimulated earlier by the other two E-RAF forms or not. This E-RAF property remained unchanged even after its association with the 4 S receptor-estrogen complex forming 5 S complex.