van de Wetering M, Clevers H
Department of Immunology, University Hospital, Utrecht, The Netherlands.
EMBO J. 1992 Aug;11(8):3039-44. doi: 10.1002/j.1460-2075.1992.tb05374.x.
The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription-regulating proteins. Two of these, the proteins encoded by the T cell-specific TCF-1 and the mammalian sex-determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF-1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF-1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T----C and A----I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence-specific binding by TCF-1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF-1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.
高迁移率族I（HMG）框被认为可介导一组新型转录调节蛋白与DNA的结合。其中两种蛋白，即由T细胞特异性基因TCF - 1编码的蛋白和哺乳动物性别决定基因SRY编码的蛋白，都带有一个对七聚体基序A/T A/T C A A A G具有特异性的单一HMG框。我们现在分析了TCF - 1和SRY的HMG框与该基序的相互作用模式。甲基化干扰足迹分析表明，两个HMG框均在小沟中与两条链上的腺嘌呤接触，而未发现与大沟鸟嘌呤的接触。TCF - 1的焦碳酸二乙酯（DEPC）乙氧基化干扰足迹分析表明，在基序的第5、6和7位不存在大沟接触。然而，在大沟的第2、3和4位观察到了乙氧基化干扰，在第1位的干扰程度较小。联合的T→C和A→I取代改变了大沟表面，但小沟保持完整，并未干扰TCF - 1和SRY的序列特异性结合。这些观察结果表明，远缘相关的TCF - 1和SRY蛋白的HMG框对七聚体基序的识别主要通过小沟中的核苷酸接触来实现。
