A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA.
A single-stranded DNA binding protein that specifically recognizes cis-acting sequences in the replication origin and transcriptional promoter region of Tetrahymena rDNA.
复制标题
一种单链 DNA 结合蛋白，特异性识别四膜虫 rDNA 复制起点和转录启动子区域中的顺式作用序列。
DOI：
10.1021/bi00014a011
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Dobbs,DL
中科院分区：
文献类型：
--
作者：
Hou,Z;Umthun,AR;Dobbs,DL
I型重复序列是嗜热四膜虫rRNA基因（rDNA）的复制起点和转录启动子区域中发现的进化上保守的序列元件。一个丰富的单链DNA结合蛋白，ssA-TIBF，特异性地与I型重复序列的富含A的链相互作用。用纯化的ssA-TIBF进行的定量结合竞争实验表明，ssA-TIBF的结合位点包括保守的33 nt元件内的序列和3 '侧翼区中的序列：将3'侧翼序列添加到I型重复寡核苷酸中使ssA-TIBF的结合亲和力增加近100倍（表观Kd = 3.0 × 10~（10）M）。ssA-TIBF结合位点的突变先前被证明是rDNA复制缺陷的决定因素，导致ssA-TIBF体外结合亲和力降低25倍。ssA-TIBF还以高亲和力结合体外转录测定确定的必需启动子区域内的I型重复序列的拷贝。ssA-TIBF对启动子重复序列的亲和力至少等于其对起始区中I型重复序列的亲和力，所述启动子重复序列在33个位置中的8个位置处不同于所述重复序列的其它拷贝。ssA-TIBF在体外的生化特性表明，它可能在体内四膜虫rDNA的复制和转录中发挥作用。
Revised Manuscript Received January 17, 1995® abstract: Type I repeat sequences are evolutionarily conserved sequence elements foundin the replication origin and transcriptional promoter region of the rRNA genes (rDNA) in Tetrahymena thermophila. An abundant single-stranded DNA bindingprotein, ssA-TIBF, specifically interacts with the A-rich strand of the Type I repeat sequence. Quantitativebinding competition experiments performed with purified ssA-TIBF demonstrate that the binding site for ssA-TIBF includes sequences both within the conserved 33 nt element and in a 3'flanking region: addition of the 3'flanking sequence to the Type I repeat oligonucleotide increases the binding affinity of ssA-TIBF by nearly 100-fold (apparent K&= 3.0 x 10~ 10 M). A mutation in the ssA-TIBF binding site previously shownto be the determinant of an rDNA replication defect in vivo results in a 25-fold decrease in ssA-TIBF binding affinity in vitro. ssA-TIBF also binds with high affinity to a copy of the Type I repeatsequence within the essential promoter region defined by in vitro transcription assays. The affinity of ssA-TIBF for the promoter repeat, which differs from other copies of the repeat at 8 out of 33 positions, is at least equal to its affinity for the Type I repeat sequences in the origin region. The biochemical properties of ssA-TIBF in vitro suggest that it could play a role in both replication and transcription of Tetrahymena rDNA in vivo.
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