John M, Leppik R, Busch S J, Granger-Schnarr M, Schnarr M
Institut de Biologie Moléculaire et Cellulaire du CNRS, Strasbourg, France.
Nucleic Acids Res. 1996 Nov 15;24(22):4487-94. doi: 10.1093/nar/24.22.4487.
We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.
我们构建了编码c-Jun和c-Fos的bZip模块序列的质粒，这些序列随后可在大肠杆菌中作为可溶性蛋白表达。在电泳迁移率变动分析和圆二色性（CD）实验中，对纯化的bZip模块与含有TRE或CRE识别位点的合成寡核苷酸的结合能力进行了测试。电泳迁移率变动分析表明，bZip Jun同二聚体和bZip Jun/Fos异二聚体与胶原酶样TRE（CTGACTCAT）结合，解离常数分别为1.4×10⁻⁷M和5×10⁻⁸M。如先前报道[帕特尔等人（1990年）《自然》347卷，572 - 575页]，DNA结合会引起蛋白质结构的显著变化。然而，我们发现DNA也会发生构象变化。这在分别含有TRE（TGACTCA）或CRE（TGACGTCA）序列的13或14个碱基对的小寡核苷酸中最为明显。在这种情况下，280nm处的DNA正CD信号增加近两倍，同时伴有3 - 4nm的蓝移。在实验误差范围内，对于含有TRE和CRE的DNA片段观察到相同的光谱变化。用非特异性DNA片段观察到的光谱变化较弱，并且在加入比特异性DNA片段所需盐浓度小得多的盐浓度后，游离DNA的信号得以恢复。令人惊讶的是，Jun/Jun同二聚体诱导的光谱变化与Jun/Fos异二聚体诱导的光谱变化并不相同。然而，在这两种情况下，正CD带的增加以及伴随的蓝移都与结合位点部分从B型向A型转变或与标准A和B结构之间的DNA构象中间体相符。
