Ikeda M, Arai K, Masai H
Department of Molecular and Developmental Biology, University of Tokyo, Japan.
Eur J Biochem. 1996 May 15;238(1):38-47. doi: 10.1111/j.1432-1033.1996.0038q.x.
South-Western screening of a glutathione-S-transferase fusion protein library constructed from the yeast Saccharomyces cerevisiae genomic DNA lead to isolation of core T-rich-strand-binding protein (CTBP) clones that bound to single-stranded DNA containing the T-rich-strand of the 11-bp core sequence of autonomously replicating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA-binding and single-stranded DNA-binding protein of S. cerevisiae. GST-CTBP1 as well as the full-length fusion protein with RBP1 (GST-RBP1) bind exclusively to the T-rich strand of the core sequence with an apparent dissociation constant of 5 nM, but not to the A-rich strand or double strand of the same sequence. Mutations within the core which reduce the number of T or C residues decrease the affinity of this protein. In keeping with this, binding of GST-CTBP1 to the core sequence is efficiently completed by poly(dT), poly(dT-dC) or poly(dC), but not by poly(dA) or poly(dG) to significant extents. Among polyribonucleic acids, GST-CTBP1 binds to poly(U) and poly(I) with greatest affinity, whereas GST-RBP1 binds to RNA in a rather non-specific manner. In no cases was affinity for RNA greater than that for DNA. Our results indicate that CTBP1/RBP1 is a polydeoxypyrimidine-binding protein of S. cerevisiae. CTBP1 contains two sets of an RNA-recognition motif (RRM) and a glutamine stretch. The binding affinity of the N-terminal or C-terminal set containing one RRM and one glutamine stretch is nearly two orders of magnitude lower than that of the wild-type CTBP1 containing both sets. The isolated N-terminal or C-terminal RRM alone (RRM1 and RRM2, respectively) is sufficient for binding nucleic acids with the binding specificity similar to that of the wild-type RRM, although the binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that the two RRMs present in CTBP1/RBP1 have differential binding affinities and that the high affinity of RRM for polydeoxypyrimidine results from synergy between two lower-affinity RRMs.
对由酿酒酵母基因组DNA构建的谷胱甘肽-S-转移酶融合蛋白文库进行South-Western筛选，导致分离出核心富含T链结合蛋白（CTBP）克隆，这些克隆与含有自主复制序列11bp核心序列富含T链的单链DNA结合。其中一个克隆CTBP1，包含先前描述的RBP1的一部分，RBP1是酿酒酵母的一种RNA结合和单链DNA结合蛋白。GST-CTBP1以及与RBP1的全长融合蛋白（GST-RBP1）仅与核心序列的富含T链结合，表观解离常数为5 nM，但不与相同序列的富含A链或双链结合。核心内减少T或C残基数量的突变会降低该蛋白的亲和力。与此一致的是，GST-CTBP1与核心序列的结合可被聚（dT）、聚（dT-dC）或聚（dC）有效完成，但在很大程度上不能被聚（dA）或聚（dG）完成。在多核糖核酸中，GST-CTBP1与聚（U）和聚（I）结合亲和力最大，而GST-RBP1以相当非特异性的方式与RNA结合。在任何情况下，对RNA的亲和力都不大于对DNA的亲和力。我们的结果表明CTBP1/RBP1是酿酒酵母的一种多脱氧嘧啶结合蛋白。CTBP1包含两组RNA识别基序（RRM）和一个谷氨酰胺延伸区。含有一个RRM和一个谷氨酰胺延伸区的N端或C端组的结合亲和力比含有两组的野生型CTBP1低近两个数量级。单独分离的N端或C端RRM（分别为RRM1和RRM2）足以以与野生型RRM相似的结合特异性结合核酸，尽管分离的RRM2的结合亲和力比RRM1低近两个数量级。我们的结果表明CTBP1/RBP1中存在的两个RRM具有不同的结合亲和力，并且RRM对多脱氧嘧啶的高亲和力是由两个低亲和力RRM之间的协同作用产生的。
