PROGESTERONE RECEPTOR-INDUCED BENDING OF ITS TARGET DNA - DISTINCT EFFECTS OF THE A-RECEPTOR AND B-RECEPTOR FORMS

We have used circular permutation and phasing electrophoretic mobility shift assays to determine the ability of the A and B forms of human p rogesterone receptor (PR) to bend target DNA. Studies were done with b aculovirus-expressed full-length receptors purified to apparent homoge neity, By circular permutation analysis, both forms of PR induced subs tantial distortions in the structure of target DNA with calculated dis tortion angles (alpha(D)) of 57 degrees for PR-A and 84 degrees for PR -B, The apparent bend centers for both forms of PR were similarly loca ted a few base pairs (-4 to -2 bp) from the middle of the progesterone response element, No differences were detected in the magnitude of di stortion or apparent bend centers when PR was bound to hormone agonist (R5020) or the antagonist RU486, Phasing analysis, which can determin e the orientation of a DNA bend, revealed that both forms of PR mediat ed directional bends toward the major groove of the DNA helix, Calcula ted directed bend angles (alpha(B)) were 40 degrees for PR-B and 31 de grees for PR-A, The chromatin high mobility group protein HMG-1, which acts as an accessory factor to enhance the binding affinity of purifi ed PR for progesterone response elements, had minimal influence on PR- mediated DNA bending, This result, taken together with the fact that H MG-1 can form a ternary complex with PR and DNA, is consistent with th e conclusion that HMG-1 facilitates PR binding by stabilizing a recept or-induced DNA conformation that is required for assembly of a high af finity PR-DNA complex, The results of this study also suggest that DNA bending may be coupled to transcriptional regulation since PR-B is ge nerally a stronger transcriptional activator than PR-A and also mediat es a larger bend in target DNA than PR-A.