Vullhorst Detlef, Buonanno Andres
Section on Molecular Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2003 Mar 7;278(10):8370-9. doi: 10.1074/jbc.M209361200. Epub 2002 Dec 9.
General transcription factor 3 (GTF3) binds specifically to the bicoid-like motif of the troponin I(slow) upstream enhancer. This motif is part of a sequence that restricts enhancer activity to slow muscle fibers. GTF3 contains multiple helix-loop-helix domains and an amino-terminal leucine zipper motif. Here we show that helix-loop-helix domain 4 is necessary and sufficient for binding the bicoid-like motif. Moreover, the affinity of this interaction is enhanced upon removal of amino-terminal sequences including domains 1 and 2, suggesting that an unmasking of the DNA binding surface may be a precondition for GTF3 to bind DNA in vivo. We have also investigated the interactions of six GTF3 splice variants of the mouse, three of which were identified in this study, with the troponin enhancer. The gamma-isoform lacking exon 23, and exons 26-28 that encode domain 6, interacted most avidly with the bicoid-like motif; the alpha- and beta- isoforms that include these exons fail to bind in gel retardation assays. We also show that GTF3 polypeptides associate with each other via the leucine zipper. We speculate that cells can generate a large number of GTF3 proteins with distinct DNA binding properties by alternative splicing and combinatorial association of GTF3 polypeptides.
通用转录因子3（GTF3）特异性结合肌钙蛋白I（慢型）上游增强子的类双胸苷基序。该基序是将增强子活性限制于慢肌纤维的序列的一部分。GTF3包含多个螺旋-环-螺旋结构域和一个氨基末端亮氨酸拉链基序。在此我们表明，螺旋-环-螺旋结构域4对于结合类双胸苷基序是必需且充分的。此外，去除包括结构域1和2在内的氨基末端序列后，这种相互作用的亲和力增强，这表明DNA结合表面的暴露可能是GTF3在体内结合DNA的前提条件。我们还研究了小鼠的六种GTF3剪接变体（其中三种是在本研究中鉴定出来的）与肌钙蛋白增强子的相互作用。缺失外显子23以及编码结构域6的外显子26 - 28的γ异构体与类双胸苷基序的相互作用最为强烈；包含这些外显子的α和β异构体在凝胶阻滞试验中未能结合。我们还表明，GTF3多肽通过亮氨酸拉链相互结合。我们推测，细胞可以通过GTF3多肽的可变剪接和组合结合产生大量具有不同DNA结合特性的GTF3蛋白。
