USE OF A SELECTION TECHNIQUE TO IDENTIFY THE DIVERSITY OF BINDING-SITES FOR THE YEAST RAP1 TRANSCRIPTION FACTOR
USE OF A SELECTION TECHNIQUE TO IDENTIFY THE DIVERSITY OF BINDING-SITES FOR THE YEAST RAP1 TRANSCRIPTION FACTOR
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DOI：
10.1093/nar/22.2.124
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发表时间：
1994-01-25
影响因子：
14.9
通讯作者：
CHAMBERS, A
中科院分区：
文献类型：
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作者：
GRAHAM, IR;CHAMBERS, A
我们使用了选择性扩增结合 (SAAB) 技术从简并寡核苷酸库中分离出酵母转录因子 RAP1 的结合位点。总共分离出 47 个序列，其中两个被证明是污染的非 RAP1 结合位点。排除这两个序列后，其余序列用于导出 RAP1 的新共有结合位点。新共识 5' A/G T A/G C A C C C A N N C C/A C C 3' 是现有共识 (4) 的重要扩展。它在 5' 端长了两个碱基对，并且在 3' 端受到明显更多的限制。与共有的 RAP1 结合位点相比，对单个 SAAB 序列中的错配组合进行分析，使我们能够更详细地分析 RAP1 结合位点的结构。结合位点可细分为三个区域；核心结合位点、5'侧翼区域和3'侧翼区域。核心结合位点由序列 5'CACCCA3' 组成，对于 RAP1 的识别至关重要。不太保守的侧翼区域并不那么重要。 RAP1 和这些区域之间的相互作用可能稳定 RAP1 和核心结合位点之间的相互作用。 SAAB 分析中分离的每个序列均用于搜索 EMBL + GenBank DNA 数据库的第 78 版。搜索确定了酵母基因启动子内 RAP1 的 102 个潜在结合位点。
We have used the technique known as selected and amplified binding (SAAB) to isolate binding sites for the yeast transcription factor RAP1 from a degenerate pool of oligonucleotides. A total of 47 sequences were isolated, of which two were shown to be contaminating non-RAP1 binding sites. After excluding these two sequences the remainder of the sequences were used to derive a new consensus binding site for RAP1. The new consensus 5' A/G T A/G C A C C C A N N C C/A C C 3' is a significant extension of the existing consensus (4). It is longer by two base pairs at the 5' end and is significantly more constrained at the 3' end. An analysis of the combinations of mis-matches in individual SAAB sequences, compared to the consensus RAP1 binding site, has allowed us to analyse the structure of the RAP1 binding site in some detail. The binding site can be sub-divided into three regions; a core binding site, a 5' flanking region and a 3' flanking region. The core binding site, consisting of the sequence 5'CACCCA3', is critical for recognition by RAP1. The less conserved flanking regions are not as important. Interactions between RAP1 and these regions probably stabilise the interaction between RAP1 and the core binding site. Each of the sequences isolated in the SAAB analysis was used to search release 78 of the EMBL + GenBank DNA data base. The searches identified 102 potential binding sites for RAP1 within promoters of yeast genes.