T7 PROTEIN-SYNTHESIS IN F-FACTOR-CONTAINING CELLS - EVIDENCE FOR AN EPISOMALLY INDUCED IMPAIRMENT OF TRANSLATION AND ITS RELATION TO AN ALTERATION IN MEMBRANE-PERMEABILITY
T7 PROTEIN-SYNTHESIS IN F-FACTOR-CONTAINING CELLS - EVIDENCE FOR AN EPISOMALLY INDUCED IMPAIRMENT OF TRANSLATION AND ITS RELATION TO AN ALTERATION IN MEMBRANE-PERMEABILITY
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DOI：
10.1128/jvi.17.1.94-105.1976
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发表时间：
1976-01-01
影响因子：
5.4
通讯作者：
MALAMY, MH
中科院分区：
文献类型：
--
作者：
BLUMBERG, DD;MABIE, CT;MALAMY, MH
含F因子的PIFA+、B+细胞的T7感染是失败的。尽管存在所有三类T7蛋白的mRNA，但只有最早的T7蛋白被合成。一个关键的问题是T7不能在PIFA+、B+细胞中发育是否是由于不能翻译T7 mRNA的晚期类别，或者如最近提出的（布里顿和Haselkorn，1975; Condit，1975），是否是由于膜渗透性的更普遍的改变。我们研究了野生型PIFA+、B+附加体和两种附加体突变（pifA−和pif B −）对体外翻译和膜通透性的影响。在体内，附加型突变允许发生部分或完全T7发育。我们证明，T7感染的PIFA+，B+细胞的无细胞蛋白质合成系统显示天然和合成mRNA的翻译速率降低3 - 5倍。此外，来自T7感染的PIFA+、B+细胞的核糖体在响应天然mRNA时结合Fmet tRNAf的能力上有缺陷。相比之下，T7感染的pifA-（PIFA-，B+）细胞的无细胞提取物保留了结合Fmet tRNAf的能力，并以正常速率翻译天然和合成mRNA。未感染的PIFA+、B+或T7感染的PIFA-、B+细胞的胰蛋白酶敏感部分可使缺陷的T7感染的PIFA+、B+核糖体恢复至完全活性。尽管这些提取物的翻译能力不同，但T7感染的PIFA+，B+和PIFA-，B+细胞显示出相同的渗透性损伤，如通过ATP从细胞损失到上清液中所测量的。附加体topfiB−的突变防止了T7感染后细胞中ATP的丢失。
T7 infection of F-factor-containing PIFA+,B+cells is abortive. In spite of the presence of mRNA for all three classes of T7 proteins, only the earliest of the T7 proteins are synthesized. A crucial question is whether the failure of T7 to develop in PIFA+,B+cells is the result of an inability to translate the late classes of T7 mRNA or, as has been recently suggested (Britton, and Haselkorn, 1975; Condit, 1975), whether it is the result of a more generalized alteration in membrane permeability. We have examined the effects of the wild-type PIFA+,B+episome and two episomal mutations (pifA−andpifB−) on in vitro translation and membrane permeability. In vivo the episomal mutations allow partial or complete T7 development to occur. We demonstrate that cell-free protein-synthesizing systems from T7-infected PIFA+,B+cells show a three- to fivefold decrease in the rate of translation of both natural and synthetic mRNA. In addition, ribosomes from T7-infected PIFA+,B+cells are defective in their ability to bind Fmet tRNAfin response to natural mRNA. By contrast, cell-free extracts from T7-infectedpifA−(PIFA−,B+) cells retain the ability to bind Fmet tRNAfand to translate natural and synthetic mRNA at normal rates. The defective T7-infected PIFA+,B+ribosomes can be restored to full activity by a trypsin-sensitive fraction from uninfected PIFA+,B+or T7-infected PIFA−,B+cells. Despite the differences in translational capacity of these extracts, both T7-infected PIFA+,B+and PIFA−,B+cells display the same permeability lesions as measured by the loss of ATP from the cells into the supernatant. Mutation of the episome topfiB−prevents the loss of ATP from the cells after T7 infection.