用于大肠杆菌的体外mRNA降解系统的开发：聚腺苷酸聚合酶I是触发降解所必需的。
Development of an in vitro mRNA decay system for Escherichia coli: poly(A) polymerase I is necessary to trigger degradation.
作者信息
Ingle C A, Kushner S R
机构信息
Department of Genetics, University of Georgia, Athens 30602-7223, USA.
出版信息
Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12926-31. doi: 10.1073/pnas.93.23.12926.
Abstract
Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from s strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) are detected on the 3' end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I-polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.
摘要
利用一种新型的大肠杆菌体外降解系统，其中多核糖体是酶和mRNA的来源，我们证明了mRNA周转过程中对聚腺苷酸聚合酶I（PAP I）的需求。只有当从携带PAP I野生型基因（pcnB+）的s菌株制备多核糖体时，添加ATP才会引发两种不同mRNA（trxA和lpp）的体外降解。这两种mRNA的相对降解率在体外和体内相似。两种mRNA上都形成了聚腺苷酸尾巴，但在成熟的23S rRNA的3'端未检测到聚腺苷酸。体外产生的聚腺苷酸尾巴的大小分布平均长度为50个核苷酸，与先前在体内报道的相当。PAP I活性仅与多核糖体相关。外源添加的PAP I不能将mRNA降解恢复到PAP I-多核糖体，这表明在体内，PAP I可能是多蛋白复合物的一部分。讨论了该体外系统用于分析大肠杆菌中mRNA降解的潜力。
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