Shafferman A, Helinski D R
J Biol Chem. 1983 Apr 10;258(7):4083-90.
The beta origin of replication of plasmid R6K, one of three active R6K origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. The nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (Stalker, D., Kolter, R., and Helinski, D. (1982) J. Mol. Biol. 161, 33-43). In this work, the sequence of the remaining portion of this 1964-bp segment was obtained. In addition to its activity as an origin of replication, this sequence also contains sufficient information for autonomous replication in Escherichia coli. A 277-bp region containing seven 22-bp direct repeats is present at one end of the beta origin segment (Stalker, D., Kolter, R., and Helinski, D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 1150-1154) while the other end contains a 140-bp sequence that includes a relaxation complex site. The 277-bp direct repeat region is required for activity of the beta origin. The start of the beta origin of replication as mapped by electron microscopy (Crosa, J. (1980) J. Biol. Chem. 255, 11075-11077) lies approximately 1000 bp away from the 277-bp region. The pi structural gene, which makes up most of the sequence between the direct repeats and the beta origin, is required in cis for beta origin activity. The pi protein also is required for beta origin activity but can be provided in trans. The nucleotide sequence just beyond the pi structural gene and within or near the start of beta origin of replication contains an open reading frame for a 151-amino acid protein. Deletions ranging from 94 bp to 1590 bp were obtained within the 1964-bp beta origin region. In every case, the deletion results in loss of origin activity even when the deleted sequence plus adjacent regions are provided in trans. These observations suggest a requirement for a specific secondary structure over an extensive region for beta origin activity.
质粒R6K的β复制起点是三个活跃的R6K复制起点之一，其活性需要一个1962碱基对（bp）序列的大部分或全部。在早期的一项研究中（斯托克，D.，科尔特，R.，和赫林斯基，D.（1982年）《分子生物学杂志》161卷，33 - 43页）确定了该功能性β起点一部分的核苷酸序列。在这项工作中，获得了这个1964 bp片段其余部分的序列。除了作为复制起点的活性外，该序列还包含在大肠杆菌中自主复制的足够信息。β起点片段的一端存在一个包含七个22 bp直接重复序列的277 bp区域（斯托克，D.，科尔特，R.，和赫林斯基，D.（1979年）《美国国家科学院院刊》76卷，1150 - 1154页），而另一端包含一个140 bp的序列，其中包括一个松弛复合体位点。277 bp直接重复区域是β起点活性所必需的。通过电子显微镜定位的β复制起点的起始位置（克罗萨，J.（1980年）《生物化学杂志》255卷，11075 - 11077页）距离277 bp区域约1000 bp。构成直接重复序列和β起点之间大部分序列的π结构基因，对于β起点活性是顺式作用必需的。π蛋白对于β起点活性也是必需的，但可以反式提供。恰好在π结构基因之后以及β复制起点起始位置之内或附近的核苷酸序列包含一个151个氨基酸蛋白质的开放阅读框。在1964 bp的β起点区域内获得了从94 bp到1590 bp不等的缺失。在每种情况下，即使缺失序列加上相邻区域以反式提供，缺失也会导致起点活性丧失。这些观察结果表明，β起点活性需要在一个广泛区域内具有特定的二级结构。
