Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phage-modified RNA polymerase.
Transcription from a bacteriophage T4 middle promoter using T4 motA protein and phage-modified RNA polymerase.
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使用 T4 motA 蛋白和噬菌体修饰的 RNA 聚合酶从噬菌体 T4 中间启动子转录。
DOI：
10.1016/s0021-9258(18)55233-9
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
D. Hinton
中科院分区：
文献类型：
--
作者：
D. Hinton
T4 中间启动子的转录需要噬菌体 T4 motA 蛋白。这些启动子在 -10 区域包含大肠杆菌启动子共有序列 (TATAAT)，但在 -30 区域包含独特序列 ((a/t)(a/t)TGCTT(t/c)A)（Guild, N.、Gayle, M.、Sweeney, R.、Hollingsworth, T.、Modeer, T. 和 Gold, L. (1988) J. Mol. Biol. 199、 241-258），在感染后约 2 分钟变得活跃，此时宿主 RNA 聚合酶已被噬菌体蛋白修饰。本文表明，motA 蛋白在体外与 T4 中间启动子结合，并且 motA 蛋白的添加允许通过 T4 修饰的 RNA 聚合酶从该启动子进行体外转录。 T4 motA 基因被克隆到多拷贝质粒中，该质粒在体内补充 T4 motA 突变体。 MotA 蛋白是从含有 motA+ 质粒的细胞中部分纯化出来的，它特异性地延迟了含有位于 uvsX (PuvsX) 上游 195 个碱基处的 T4 中间启动子的寡聚物的电泳迁移率。仅当添加含有 motA 蛋白的级分时，在 T4 中间基因表达期间从感染细胞中分离出的 RNA 聚合酶才支持 PuvsX 的体外转录。相反，未修饰的宿主RNA聚合酶催化PuvsX合成少量RNA，但这种合成不依赖于motA。因此，这里描述的体外转录系统为调节 T4 中间基因表达所需的噬菌体和宿主因子的详细研究提供了基础。
The bacteriophage T4 motA protein is required for transcription from T4 middle promoters. These promoters, which contain the Escherichia coli promoter consensus sequence at the -10 region (TATAAT) but a unique sequence centered at -30 ((a/t)(a/t)TGCTT(t/c)A) (Guild, N., Gayle, M., Sweeney, R., Hollingsworth, T., Modeer, T., and Gold, L. (1988) J. Mol. Biol. 199, 241-258), become active about 2 min after infection, a time when the host RNA polymerase has been modified by phage proteins. This paper shows that motA protein binds to a T4 middle promoter in vitro and that the addition of the motA protein allows in vitro transcription from this promoter by T4-modified RNA polymerase. The T4 motA gene was cloned into a multicopy plasmid that complemented T4 motA mutants in vivo. MotA protein, partially purified from cells containing a motA+ plasmid, specifically retarded the electrophoretic mobility of an oligomer containing the T4 middle promoter located 195 bases upstream of uvsX (PuvsX). RNA polymerase isolated from infected cells during T4 middle gene expression supported in vitro transcription from PuvsX only when fractions containing the motA protein were added. In contrast, unmodified host RNA polymerase catalyzed the synthesis of minor amounts of RNA from PuvsX, but this synthesis was not motA dependent. Thus, the in vitro transcription system described here provides the basis for a detailed study of the phage and host factors needed to regulate T4 middle gene expression.