Progesterone receptor-induced bending of its target DNA: distinct effects of the A 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 progesterone receptor (PR) to bend target DNA. Studies were done with baculovirus-expressed full-length receptors purified to apparent homogeneity. By circular permutation analysis, both forms of PR induced substantial distortions in the structure of target DNA with calculated distortion 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 located a few base pairs (-4 to -2 bp) from the middle of the progesterone response element. No differences were detected in the magnitude of distortion or apparent bend centers when PR was bound to hormone agonist (R5020) or the antagonist RU486. Phasing analysis, which can determine the orientation of a DNA bend, revealed that both forms of PR mediated directional bends toward the major groove of the DNA helix. Calculated directed bend angles (alpha B) were 40 degrees for PR-B and 31 degrees for PR-A. The chromatin high mobility group protein HMG-1, which acts as an accessory factor to enhance the binding affinity of purified PR for progesterone response elements, had minimal influence on PR-mediated DNA bending. This result, taken together with the fact that HMG-1 can form a ternary complex with PR and DNA, is consistent with the conclusion that HMG-1 facilitates PR binding by stabilizing a receptor-induced DNA conformation that is required for assembly of a high affinity PR-DNA complex. The results of this study also suggest that DNA bending may be coupled to transcriptional regulation since PR-B is generally a stronger transcriptional activator than PR-A and also mediates a larger bend in target DNA than PR-A.
Prendergast
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Department of Pathology, University of Colorado Health Sciences Center Denver 80262, USA.
Pan
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Edwards
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D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D015735:Mifepristone / Q000494:pharmacology; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D011980:Receptors, Progesterone / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014158:Transcription, Genetic; D014162:Transfection
D015342:DNA Probes; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009838:Oligodeoxyribonucleotides; D011980:Receptors, Progesterone; D011994:Recombinant Proteins; C097314:progesterone receptor B; D015735:Mifepristone; D004247:DNA