Türkel S, Farabaugh P J
Department of Biological Sciences, University of Maryland, Catonsville 21228.
Mol Cell Biol. 1993 Apr;13(4):2091-103. doi: 10.1128/mcb.13.4.2091-2103.1993.
Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.
酿酒酵母Ty2 - 917反转录转座子的转录受到一组复杂的正负调控元件的调节，其中包括位于第一个开放阅读框TYA2内的一个负调控区域。该负调控区域包含三个下游抑制位点（DRSI、DRSII和DRSIII）。此外，该负调控区域还包含至少两个下游激活位点（DASs）。本文关注DASI的特性。一个36bp的DASI寡核苷酸作为一个自主转录激活位点，包含两个激活所必需的序列元件。我们发现这些位点在体外能结合转录激活蛋白GCN4，并且它们在体内的活性对细胞中GCN4的水平有响应。我们将这两个位点称为GCN4结合位点（GBS1和GBS2）。GBS1是一个高亲和力的GCN4结合位点（解离常数，30℃时约为25 nM），以大约与共有UASGCN4相同的亲和力结合GCN4，尽管GBS1与回文共有位点的右半部分有两个差异。GBS2与共有序列的差异更大，结合GCN4的亲和力约低20倍。DASI的第13至36个核苷酸与DRSII重叠。由于DRSII是一个转录抑制位点，我们测试了DASI是否包含抑制元件。我们在GBS2两侧鉴定出两个位点，它们都能抑制由共有GCN4特异性上游激活位点（UASGCN4）激活的转录。其中一个在紧邻DASI的12bp中重复出现。因此，在Ty2 - 917的一个48bp区域内散布着两个正转录调节因子和三个负转录调节因子。该区域的净效应必定取决于结合在这些位点上的蛋白质之间的相互作用，这可能包括它们对结合位点的竞争，以及这些蛋白质活性的生理调控。
