Lee E H, Masai H, Allen G C, Kornberg A
Department of Biochemistry, Beckman Center, Stanford University, CA 94305-5307.
Proc Natl Acad Sci U S A. 1990 Jun;87(12):4620-4. doi: 10.1073/pnas.87.12.4620.
The Escherichia coli gene encoding protein n' has been isolated and named priA for primosomal protein A. Protein n' is absolutely required for the conversion of single-stranded phi X174 DNA to the duplex replicative form in an in vitro-reconstituted system. The gene maps to 88.7 minutes on the chromosome adjacent to the cytR locus. Soluble protein extracts from cells harboring the priA gene on a multicopy plasmid contained 45-fold more n' replication activity than wild-type extracts. Enhanced overproduction of greater than 1000-fold was achieved by replacing the natural Shine-Dalgarno sequence with that of the phage T7 phi 10 gene and placing this priA under the control of the T7 phage promoter and RNA polymerase. The priA sequence reveals a 732-amino acid open reading frame and a nucleotide-binding consensus site consistent with the size and ATPase activity of the purified protein. The gene for protein n has been named priB and the putative gene for protein n", priC.
编码蛋白n'的大肠杆菌基因已被分离出来，并命名为priA，即引发体蛋白A。在体外重组系统中，将单链φX174 DNA转化为双链复制形式绝对需要蛋白n'。该基因定位于染色体上88.7分钟处，与cytR基因座相邻。在多拷贝质粒上携带priA基因的细胞的可溶性蛋白提取物中，n'复制活性比野生型提取物高45倍。通过用噬菌体T7 φ10基因的Shine-Dalgarno序列取代天然序列，并将该priA置于T7噬菌体启动子和RNA聚合酶的控制之下，实现了超过1000倍的增强过量表达。priA序列揭示了一个732个氨基酸的开放阅读框和一个与纯化蛋白的大小和ATP酶活性一致的核苷酸结合共有位点。蛋白n的基因已被命名为priB，而蛋白n''的推定基因则为priC。
