BACTERIOPHAGE T4 GENE EXPRESSION

正常的细胞发育需要 RNA聚合酶与多种转录调控因子 活动我们研究了噬菌体T4中的转录， 启动子，其中E.杆菌RNA 聚合酶需要噬菌体编码的转录激活因子， MotA蛋白和T4共激活因子AsiA蛋白。该系统 是一个简单的模型，用于研究因素如何改变 聚合酶的特异性。E.大肠杆菌RNA聚合酶全酶， 由4个子单元的核心加上西格玛因子组成， 识别特定的启动子序列。在过去的一年里，我们 一直在研究613个氨基酸中的突变是如何发生的， sigma-70是E. coli，影响转录。 先前的工作表明， sigma-70（区域4）需要与-35区域相互作用 细菌启动子和特定的细菌激活剂。我们 已经证明缺乏该结构域的sigma-70蛋白 无法绑定到MotA或AsiA，并且不支持MotA/AsiA 激活转录。这些结果表明，MotA/AsiA T4中间启动子的激活是通过 噬菌体蛋白与σ-70区域4的相互作用。一 先前对一组17个sigma-70错义突变体的研究 揭示了特定的C-末端氨基酸是必需的， 与特定的细菌激活剂相互作用。但我们 发现这些突变体中的每一个都支持中间转录， 体内，表明MotA/AsiA与sigma-70的相互作用 不同于细菌系统中的活化剂。虽然 sigma-70识别启动子DNA，当它存在于 聚合酶全酶，sigma-70本身不结合DNA。它 已经表明σ-70区域的前100个氨基酸 1.1)负责单独通过σ-70抑制DNA结合。 我们已经研究了区域1.1的作用时，sigma- 70是 存在于全酶中。我们发现， 从特定启动子转录的全酶被调节， 区域1.1.一种缺乏1.1区的sigma-70全酶， 活性低于全酶，具有全长σ-70， 从强启动子转录。然而，它更容易使用 弱启动子，命名为Pminor。我们已经证明 缺少区域1.1的全酶在Pminor处更有活性，因为 它与该促进剂迅速形成稳定的复合物， 而具有全长σ-70的全酶则没有。我们还 发现T4辅激活因子AsiA更容易与sigma-70结合， 缺少1.1区的基因比与全长σ-70结合的基因要少。我们 结果表明，sigma-70的N-末端区域（区域1.1） 调节sigma-70的C末端区域的可及性 （区域4）对于启动子DNA和调节因子两者。

Normal cell development requires the interaction of RNA polymerase with many factors that regulate transcriptional activity. We study transcription from bacteriophage T4 middle promoters in which the initiation of transcription by E. coli RNA polymerase requires the phage-encoded transcriptional activator, MotA protein, and the T4 co-activator, AsiA protein. This system is a simple model for examining how factors can change the specificity of a polymerase. E. coli RNA polymerase holoenzyme is composed of a core of 4 subunits plus a sigma factor that recognizes specific promoter sequences. In the past year, we have been investigating how mutations within the 613 amino acids of sigma-70, the major sigma subunit in E. coli, affect transcription. Previous work has shown that the far C-terminal domain of sigma-70 (region 4) is required to interact both with the -35 region of bacterial promoters and with particular bacterial activators. We have demonstrated that sigma-70 protein lacking this domain cannot bind to MotA or to AsiA and does not support MotA/AsiA activated transcription. These results suggest that MotA/AsiA activation of T4 middle promoters is achieved through the interaction of the phage proteins with region 4 of sigma-70. A previous study of a set of seventeen sigma-70 missense mutants revealed that specific C-terminal amino acids are required for interacting with particular bacterial activators. However, we have found that each of these mutants supports middle transcription in vivo, suggesting that the MotA/AsiA interaction with sigma-70 differs from that of activators in the bacterial systems. Although sigma-70 recognizes promoter DNA when it is present in polymerase holoenzyme, sigma-70 by itself does not bind DNA. It has been shown that the first 100 amino acids of sigma-70 region 1.1) are responsible for inhibiting DNA binding by sigma-70 alone. We have investigated the role of region 1.1 when sigma- 70 is present in holoenzyme. We have found that the ability of holoenzyme to transcribe from specific promoters is modulated by region 1.1. A holoenzyme with a sigma-70 that lacks region 1.1 is less active than holoenzyme with a full length sigma-70 in transcribing from strong promoters. However, it more readily uses a weak promoter, designated Pminor. We have shown that holoenzyme that lacks region 1.1 is more active at Pminor because it rapidly forms stable complexes with this promoter while holoenzyme with a full length sigma-70 does not. We have also found that the T4 coactivator AsiA binds more readily to sigma-70 that lacks region 1.1 than it binds to full length sigma-70. Our results suggest that the N-terminal region of sigma-70 (region 1.1) modulates the accessibility of the C- terminal region of sigma-70 (region 4) both for promoter DNA and for regulatory factors.