1. Figure 3. Kinetics effects of the C domain on ER–EREc binding
Figure 3. Kinetics effects of the C domain on ER–EREc binding. (A) Association kinetics (on-rate) of rtER and rtER(hC) on EREc. Equal amounts of in vitro translated rtER or rtER(hC) were incubated with poly dI-dC for 15 min, then ³²P-labeled EREc was added and the reaction mixture was further incubated for 0, 5, 10, 15, 20 and 25 min. The samples were then loaded on a 5% polyacrylamide gel. (B) Dissociation kinetics (off-rate) of rtER and rtER(hC) on EREc. The reactions were incubated with poly dI-dC for 15 min, then the probe was added for 25 min. A 3-fold molar excess of cold EREc was added and reaction samples were loaded on a 5% polyacrylamide gel after 0, 5, 10, 20 and 30 min incubation. ER–EREc complexes were quantitated using an Instantimager (Packard). Results are represented as the means ± SEM from three or four separate experiments. A representative experiment is showed on top of the graph for each receptor.
From: The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function
Nucleic Acids Res. 2000;28(14): doi: /nar/
Nucleic Acids Res |

2. Figure 1. Transactivation analysis of wild-type rtER, hER and chimeric ERs in yeast. (A) Schematic representation of rtER, hER and the chimeric ERs rtER(hAB), rtER(hCD), rtER(hEF), rtER(hC), rtER(hD) and hER(rtC). The regions containing the DBD and HBD are shown in full segment and hatched from right to left, respectively. Percentages indicate amino acid identity between hER and rtER. (B) Aliquots of 10 µg of nuclear extracts of untransformed (-ER) or indicated receptor transformed yeast were resolved on a 10% polyacrylamide gel and electrotransfered to a nylon membrane. Western blots were performed using a specific anti-rtER antibody-Ab₃ (left) or an anti-hER antibody-H222 (right). The position of the molecular weight marker (66 KDa) is indicated at the left side. For the transactivation analyses, yeast strain BJ-ECZ containing the reporter gene 2ERE-CYC1-lacZ integrated in the genome and expressing either no receptor (-ER), rtER, hER, rtER(hAB), rtER(hCD), rtER(hEF) (C), rtER(hC), rtER(hD) or hER(rtC) (D) were grown in selective medium containing an increasing concentration of E₂. The β-galactosidase activity was measured in yeast extracts. Values represent the means ± SEM from at least four separate experiments.
From: The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function
Nucleic Acids Res. 2000;28(14): doi: /nar/
Nucleic Acids Res |

3. Figure 2. Examination of the relative DNA-binding affinity of wild-type and chimeric ERs to a consensus ERE. (A) rtER, rtER(hC), hER and hER(rtC) were in vitro translated in reticulocyte lysate in the presence of ³⁵S-labeled methionine/cysteine, run on a 10% SDS–PAGE and autoradiographed. (B) Binding reactions containing 4 µl of in vitro translated ER were incubated with increasing amounts of consensus ERE before the addition of 0.08 pmol of ³²P-labeled EREc. The molar excess of unlabeled ERE added was 0-fold (lanes 1, 8, 15 and 22), 1-fold (lanes 2, 9, 16 and 23), 2-fold (lanes 3, 10, 17 and 24), 5-fold (lanes 4, 11, 18 and 25), 10-fold (lanes 5, 12, 19 and 26), 20-fold (lanes 6, 13, 20 and 27) and 50-fold (lanes 7, 14, 21 and 28). Analysis of protein binding by gel retardation was accomplished as described in Materials and Methods. The arrows 1 and 2–3 show the specific ER–ERE complexes for rtER and rtER(hC), and hER and hER(rtC), respectively. The figure shows the result of a representative gel retardation out of three. Gels were then scanned with an Instantimager (Packard). The relative ER amount bound to the ERE without any competition (lanes 1, 8, 15 and 22) was determined (C) and the relative binding (%) of the different receptors were plotted against the molar excess of competitor EREc (D).
From: The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function
Nucleic Acids Res. 2000;28(14): doi: /nar/
Nucleic Acids Res |

4. Figure 4. Transcriptional activation of reporter genes containing one or three EREs, by wild-type and chimeric ERs. Yeast strain FL100 containing either reporter plasmid pLRΔ21-U1ERE (A), or pLRΔ21-U3ERE (B), expressing no receptor (-ER), rtER, hER, rtER(hAB), rtER(hCD), rtER(hEF), rtER(hC), rtER(hD) or hER(rtC) were grown in the absence or presence of 10^–8 or 10^–6 M E₂. The β-galactosidase activity was measured in yeast extracts. Values represent the means ± SEM from at least four separate experiments.
From: The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function
Nucleic Acids Res. 2000;28(14): doi: /nar/
Nucleic Acids Res |

5. Figure 5. Transactivation analysis of wild-type rtER, hER and mutated ERs in yeast. (A) Schematic representation of the two zinc fingers present in the rtER DBD. The closed circle amino acids represent those of hER different from those of rtER. Conserved cysteine residues that function to coordinate zinc ions are in bold. Helix 1 contains P-box amino acid residues that provided deoxynucleotide contacts and fit into the major groove of the DNA helix. The three P-box residues, indicated by closed circles, are involved in binding site discrimination. The five amino acid residues of the D-box are involved in the dimerization between the DBD of homodimeric ERs. The arrowhead shows the arginine we added or removed in rtER or hER, respectively, to create rtER-M1 and hER-M1, respectively. (B) Yeast strains BJ2168 containing the reporter plasmid pLRΔ21-U1ERE and expressing rtER, rtER-M1, hER or hER-M1 were grown in selective medium in the presence or absence of 10^–9 or 10^–8 M E₂. The β-galactosidase activity was measured in yeast extracts. Values represent the means ± SEM from at least seven separate experiments.
From: The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function
Nucleic Acids Res. 2000;28(14): doi: /nar/
Nucleic Acids Res |