Biochemical studies of yeast replication or gin binding protein sand initiate complexes

从酵母酵母基因组DNA构建谷胱甘肽-s转移酶融合蛋白文库的西南筛选，分离出核心富t链结合蛋白（CTBP）克隆，该克隆与含有自主复制序列11个碱基对核心序列中富t链的单链DNA结合。其中一个克隆CTBP1含有先前描述的RBP1的一部分，RBP1是酿酒酵母的RNA和单链DNA结合蛋白。GST-CTBP1融合蛋白仅与核心序列的富t链结合，表观解离常数为5*10^<-9> M，而不与同一序列的富a链或双链结合。核心内的突变减少了T或C残基的数量，降低了对该蛋白质的亲和力。与此相一致的是，与核心序列的结合被poly [dT]或ply [dT-dC]以及poly [dC]有效地竞争，尽管程度较小，但不会被poly [dA]或…更多的poly [dG]竞争。在聚核糖核酸中，它与聚[U]和聚[T]结合的亲和力分别与聚[dT]和聚[dC]相似但不大于。结果表明，CTBP1是酿酒酵母的聚脱氧嘧啶结合蛋白。CTBP1包含两组RNA识别基序（RRM）和谷氨酰胺延伸。含有一个RRM和一个谷氨酰胺拉伸的n端或c端集的结合亲和力比同时含有两个RRM和谷氨酰胺拉伸的“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）足以结合核酸，其结合特异性与“野生型”相似，尽管分离的RRM2的结合亲和力比RRM1低近两个数量级。我们的研究结果表明，CTBP1/RBP1中存在的两个RRMs具有不同的结合亲和力，其对聚脱氧嘧啶的高亲和力是两个低亲和力RRMs之间协同作用的结果。少

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 base-pair core sequence of autonomously replicating sequences. One of these clones, CTBP1, contains a portion of previously described RBP1 which is an RNA- and single-stranded DNA binding protein of Saccharomyces cerevisiae. GST-CTBP1 fusion protein binds exclusively to the T-rich strand of the core sequence with apparent dissociation constant of 5*10^<-9> M 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 to this protein. In keeping with this, binding to the core sequence is efficiently competed by poly [dT] or ply [dT-dC] as well as by poly [dC] albeit to a lesser extent, but not by poly [dA] or … More poly [dG] to any significant extent. Among polyribonucleic acids, it binds to poly [U] and poly [T] with affinity similar to but not greater than that of poly [dT] and poly [dC], respectively. Our results indicate that CTBP1 is a polydeoxypyrimidine binding protein of Saccharomyces 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", although binding affinity of the isolated RRM2 is nearly two orders of magnitude lower than that of RRM1. Our results indicate that two RRMs present in CTBP1/RBP1 have differential binding affinity and that its high affinity to polydeoxypyrimidine results from synergy between two lower affinity RRMs. Less