The yeast protein encoded by PUB1 binds T-rich single stranded DNA
The yeast protein encoded by PUB1 binds T-rich single stranded DNA
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PUB1编码的酵母蛋白结合富含T的单链DNA
DOI：
10.1093/nar/22.1.32
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发表时间：
1994
影响因子：
14.9
通讯作者：
S. Gasser
中科院分区：
文献类型：
--
作者：
M. Cockell;S. Frutiger;G. Hughes;S. Gasser
我们已经表征了酵母中的结合活性，其识别酵母ARS共识元件的富含T的链，并将其中两个纯化至均一。一种（ACBP-60）在细胞核和全细胞提取物中均可检测到，而另一种（ACBP-67）仅在通过肝素-琼脂糖色谱分离提取物后才明显。在细胞核提取物中检测到的主要结合活性在序列特异性DNA亲和柱上纯化为表观迁移率为60 kDa的单一多肽（ACBP-60）。这种蛋白质与细胞核共分级分离，每个细胞存在数千个拷贝，对ARS共有区的富含T的单链的Kd在10（-9）和10（-10）M之间。用简单核酸聚合物进行的竞争研究表明，ACBP-60对聚dT 30的亲和力略高于对含有ARS 307的富T链的30 nt寡聚体的亲和力，对聚rU的亲和力约高10倍。纯化的p60的内部序列信息揭示了与编码多聚尿苷酸结合蛋白的基因PUB 1和RNP 1的开放阅读框的同一性。第二种结合活性ACBP-67也特异性结合ARS共有序列的富含T的单链，但亲和力比ACBP-60低得多。肽序列显示67 kDa蛋白与酵母中的主要polyA结合蛋白PAB 1相同。
We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.