CONTROL OF INITIATION FREQUENCY OF COLE2 REPLICATION

ColE2 Rep蛋白在许多必需的起始蛋白中是独特的，因为它是质粒特异性引发酶，特异性用于通过DNA聚合酶I起始处的DNA复制。Rep蛋白的表达通过小反义RNA（RNAi）的负调控保持恒定。RNAl与Rep mRNA的5'非翻译区完全互补。由于Rep蛋白是反式作用的，因此如何保持起始频率恒定并不那么明显。对携带缺失或单碱基取代的ColE 2起源的各种衍生物的分析表明，该区域可分为三个亚区：一个对于Rep蛋白的稳定结合是重要的，另一个对于结合和复制是重要的，另一个对于复制是重要的但显然对于结合不是重要的。与野生型质粒相比，在第一区域中携带缺失的自主复制质粒的转化频率较低，然而它们在宿主细菌中的拷贝数较高。Rep蛋白可能通过稳定地结合到起始点来抑制新复制的子分子的过度复制或再复制。Rep mRNA起始密码子上游的5'端非翻译区和编码区中靠近起始密码子的另一个区域对于Rep蛋白的高效表达是重要的。在存在或不存在RNAi的情况下用各种RNA酶部分消化表明，RNAi与Rep mRNA的结合引起含有起始密码子的区域的二级结构的变化。突变质粒携带的Rep mRNA的起始密码子的上游和下游区域的碱基取代已被分离。一些突变消除或减少Rep蛋白的表达。

The ColE2 Rep protein is unique among many essential initiator proteins in that it is a plasmid-specified primase specific for initiation of DNA replication in the origin by DNA polymerase I.Expression of the Rep protein is kept constant through negative regulation by a small antisense RNA (RNAI). RNAl is entirely complementary to the 5' nontranslated region of the Rep mRNA.Since the Rep protein is trans-acting, it is not so obvious how the initiation frequency can be kept constant.Analyses of various derivatives of the ColE2 origin carrying deletions or single-base substitutions showed that the region may be divided into three subregions : one important for stable binding of the Rep protein, another important for binding and for replication and another important for replication but apparently not for binding. Transformation frequency of the autonomously replicating plasmids carrying deletions in the first region is lower and nevertheless the copy numbers of them in hostbacteria are higher as compared with the wild-type plasmid. The Rep protein might inhibit over-replication or re-replication of newly replicated daughter molecules by stable binding to the origin. This might be important to keep the initiation frequency at a constant level.The 5' nontranslated region of the Rep mRNA far upstream of the initiation codon and another region in the coding region near the initiation codon are important for efficient expression of the Rep protein. Partial digestion with various RNases in the presence or absence of RNAI suggested that binding of RNAI to the Rep mRNA causes changes in the secondary structure of the region containing the initiation codon. Mutant plasmids carrying base substitutions in the upstream and downstream regions of the initiation codon of the Rep mRNA have been isolated. Some of the mutations abolished or decreased expression of the Rep protein.