Ikeda R A, Chang L L, Warshamana G S
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400.
Biochemistry. 1993 Sep 7;32(35):9115-24. doi: 10.1021/bi00086a016.
The compatible plasmids pKGP1-1 and pCM-X# will confer chloramphenicol resistance to Escherichia coli harboring the two plasmids if the T7 RNA polymerase produced from pKGP1-1 can recognize the T7 promoter carried on pCM-X# and transcribe the CAT gene that is cloned behind the promoter [Ikeda et al. (1992) Biochemistry 31, 9073-9080]. When E. coli harbor pKGP1-1 and a pCM-X# plasmid that carries a point mutation in the T7 promoter that destroys promoter activity (an inactive pCM-X#), the T7 RNA polymerase will not utilize the T7 promoter point mutant, will not produce CAT, and will not induce chloramphenicol resistance. The selection of mutants of T7 RNA polymerase that exhibit altered promoter recognition was pursued by randomly mutagenizing pKGP1-1 with aqueous hydroxylamine, cotransforming E. coli with the mutagenized pKGP1-1 and a mixture of seven different inactive pCM-X# plasmids, and isolating and characterizing the RNA polymerase that was present in those colonies that exhibited chloramphenicol resistance. It was established that E. coli harboring the mutant plasmid pKGP-HA1mut4 and an inactive pCM-X# are chloramphenicol-resistant and that the mutation responsible for the expression of CAT from the inactive pCM-X# plasmid is a G to A transition at nucleotide 664 of T7 gene 1 that converts glutamic acid (222) to lysine. Apparently this mutation expands the range of T7 promoter sequences that can be utilized by the enzyme. The mutant T7 RNA polymerase, GP1(Lys222), utilizes all seven inactive T7 promoter point mutants more efficiently than wild-type T7 RNA polymerase both in vivo and in vitro. Furthermore, the correlation of in vivo and in vitro promoter utilization suggests that the restoration of chloramphenicol resistance in the cotransformed E. coli results from the ability of GP1(Lys222) to initiate transcription from T7 promoter point mutants that are normally inactive.
如果由pKGP1-1产生的T7 RNA聚合酶能够识别pCM-X#上携带的T7启动子并转录克隆在该启动子后面的CAT基因，那么相容质粒pKGP1-1和pCM-X#将赋予携带这两种质粒的大肠杆菌氯霉素抗性[池田等人（1992年）《生物化学》31卷，9073 - 9080页]。当大肠杆菌携带pKGP1-1和一个在T7启动子中携带点突变从而破坏启动子活性的pCM-X#质粒（一个无活性的pCM-X#）时，T7 RNA聚合酶将不会利用该T7启动子点突变体，不会产生CAT，也不会诱导氯霉素抗性。通过用水性羟胺对pKGP1-1进行随机诱变、将诱变后的pKGP1-1与七种不同的无活性pCM-X#质粒混合物共转化大肠杆菌，以及分离和鉴定那些表现出氯霉素抗性的菌落中存在的RNA聚合酶，来筛选表现出改变的启动子识别能力的T7 RNA聚合酶突变体。已确定携带突变体质粒pKGP-HA1mut4和无活性pCM-X#的大肠杆菌对氯霉素具有抗性，并且负责从无活性pCM-X#质粒表达CAT的突变是T7基因1的第664位核苷酸处的G到A转换，该转换将谷氨酸（222）转变为赖氨酸。显然，这种突变扩展了该酶可以利用的T7启动子序列范围。突变型T7 RNA聚合酶GP1(Lys222)在体内和体外比野生型T7 RNA聚合酶更有效地利用所有七种无活性T7启动子点突变体。此外，体内和体外启动子利用的相关性表明，共转化大肠杆菌中氯霉素抗性的恢复是由于GP1(Lys222)能够从通常无活性的T7启动子点突变体启动转录。
