Saikumar P, Murali R, Reddy E P
Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104.
Proc Natl Acad Sci U S A. 1990 Nov;87(21):8452-6. doi: 10.1073/pnas.87.21.8452.
The c-myb protooncogene codes for a sequence-specific DNA-binding protein that appears to act as a transcriptional regulator and is highly conserved through evolution. The DNA-binding domain of Myb has been shown to contain three imperfectly conserved repeats of 52 amino acids that constitute the amino-terminal end. Within each repeat, there are three tryptophans that are separated by 18 or 19 amino acids and are flanked by basic amino acids. To determine the role of tryptophans and the flanking basic amino acids in the DNA-binding activity of Myb proteins, we have selectively mutagenized individual tryptophans as well as some of the amino acid residues that flank these tryptophans. Replacement of these tryptophans with glycine, proline, or arginine abolished the DNA-binding activity whereas replacement with other aromatic amino acids or leucine or alanine did not appreciably affect this activity. On the other hand the replacement of two amino acids, asparagine and lysine, that flank the last tryptophan with acidic amino acids completely abolished their DNA-binding activity. These results are consistent with a model we present in which the tryptophans form a hydrophobic scaffold that plays a crucial role in maintaining the helix-turn-helix structure of the DNA binding domain. Basic and polar amino acids adjacent to these tryptophans seem to participate directly in DNA binding.
c-myb原癌基因编码一种序列特异性DNA结合蛋白，该蛋白似乎作为转录调节因子起作用，并且在进化过程中高度保守。已证明Myb的DNA结合结构域包含三个由52个氨基酸组成的不完全保守重复序列，构成氨基末端。在每个重复序列中，有三个色氨酸，它们被18或19个氨基酸隔开，并两侧是碱性氨基酸。为了确定色氨酸和侧翼碱性氨基酸在Myb蛋白DNA结合活性中的作用，我们选择性地诱变了单个色氨酸以及这些色氨酸侧翼的一些氨基酸残基。用甘氨酸、脯氨酸或精氨酸取代这些色氨酸会消除DNA结合活性，而用其他芳香族氨基酸、亮氨酸或丙氨酸取代则不会明显影响该活性。另一方面，用酸性氨基酸取代最后一个色氨酸侧翼的两个氨基酸天冬酰胺和赖氨酸，完全消除了它们的DNA结合活性。这些结果与我们提出的一个模型一致，在该模型中色氨酸形成一个疏水支架，在维持DNA结合结构域的螺旋-转角-螺旋结构中起关键作用。与这些色氨酸相邻的碱性和极性氨基酸似乎直接参与DNA结合。
