Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution.
Cooperative binding of initiator protein to replication origin conferred by single amino acid substitution.
复制标题
通过单个氨基酸取代赋予起始蛋白与复制起点的协同结合。
DOI：
10.1093/nar/22.20.4211
复制
发表时间：
1994
影响因子：
14.9
通讯作者：
Levchenko,I
中科院分区：
文献类型：
--
作者：
Filutowicz,M;York,D;Levchenko,I
质粒R6 K的复制起始蛋白π在π起点结合7个22 bp的同向重复序列（DR）。γ蛋白还与其自身基因pir的操纵子中的反向重复序列（IR）结合，该基因位于γ起始序列之外。设计遗传系统以选择区分IR和OR的π蛋白突变体（约克（et al.，基因（Amst.）116，7-12，1992;约克和Filutowicz，生物化学杂志，268，21854-21861，1993）。从这种选择中分离出突变体π S87 N蛋白，其在抑制pir基因的表达方面是缺陷的，因为它不能在pir基因操纵子处结合IR。值得注意的是，我们发现π S87 N在wt π独立结合的条件下协同结合DR。此外，π S87 N作为体内复制起始剂在与wt π相同水平下提供时更有活性。定量结合试验表明，wt π和TTS 87 N都以相似的亲和力（Kd= 0.3×10− 12 M）结合含有单个DR单元的DNA片段。因此，π TS 87 N的协同性最有可能通过改变在相邻DR单元处结合的原聚体之间的相互作用来实现。
The replication initiator protein π of plasmid R6K binds seven 22 bp direct repeats (DR) in the π origin. The γ protein also binds to an inverted repeat (IR) in the operator of its own gene,pir, which lies outside the γ origin sequences. A genetic system was devised to select for π protein mutants which discriminate between IR and OR (York (et al., Gene (Amst.) 116, 7–12, 1992; York and Filutowicz,J. Biol. Chem.268, 21854–21861, 1993). From this selection the mutant πS87N protein was isolated which is deficient in repressing thepirgene's expression because it cannot bind to IR at the pir gene operator. Remarkably, we discovered that πS87N binds to DR cooperatively under conditions where wt π binds independently. Moreover, the πS87N is more active as a replication initiatorin vivowhen supplied at the same level as wt π. Quantitative binding assays showed that both wt π and TTS87N bind a DNA fragment containing a single DR unit with a similar affinity (Kd= 0.3×10−12M). Thus, cooperativity of πTS87N is most likely achieved through altered interactions between protomers bound at adjacent DR units.