Bacteriophage T4 Gene Expression

正常的细胞发育需要转录启动和激活的调节，以便在适当的时间表达适当的基因。我们使用一个简单的原核系统来研究转录启动的调控：E.coliRNA聚合酶，一个由核心(2个α、β和β‘)和一个西格玛特异性因子组成的五个亚基复合体。Sigma70因子指定来自启动子的转录，这些启动子负责营养生长过程中基础基因的表达。当Sigma70存在于聚合酶中时，其两个结构域2.4(内部区域)和4.2(C末端区域)分别与启动子DNA的-10和-35区域内的序列相互作用。然而，在游离的sigma70中，DNA结合受域1.1的阻止，结构域1.1是蛋白质的N-末端区域。为了研究结构域1.1对存在于聚合酶中的sigma70的DNA结合特性的影响，我们使用不同的启动子在体外转录实验中比较了有和没有结构域1.1的聚合酶，并在评估稳定的转录前复合体的形成和衰退的实验中进行了比较。我们发现，根据启动子的不同，结构域1.1的缺失可以通过影响稳定的转录前复合体的形成来促进或抑制转录的启动。然而，结构域1.1一旦形成，不会影响这些络合物的稳定性。对于含有结构域1.1的聚合酶，稳定的复合体形成的效率与启动子的-10和-35区域与理想的sigma70识别序列匹配的程度有关。然而，当没有结构域1.1时，具有这种匹配就变得不那么重要了，以确定如何有效地制造稳定的络合物。我们的工作首次证明，当Sigma70存在于聚合酶中时，结构域1.1具有调节作用。我们认为，结构域1.1通过限制聚合酶主要通过ITS-10和-35区域与规范序列的适合性来评估启动子来影响启动子的启动。噬菌体T4蛋白MoTA是转录激活子，ASIA是共激活子，它们与RNA聚合酶的sigma70亚基相互作用，将其启动子专一性从宿主启动子转换为T4中间启动子。中间启动子含有-10处的sigma70识别序列，但缺少典型的-35序列。相反，它们包含一个以-30为中心的9BP基序(Mota框)。Mota结合Mota框并与sigma70相互作用。我们发现MoTA(MoTA NTD)的N-末端部分被认为包括激活结构域，在大肠杆菌2-杂交试验中与Sigma70的C-末端区域相互作用。Sigma70的C端17个残基被另一个Sigma的类似残基Sigma38替换，或者位于Sigma70‘S 613氨基酸末端的Sigma70突变R608C在本实验中是有缺陷的。这些结果表明，Sigma70的远C末端区域对MoTA/Sigma70的相互作用是重要的。我们还发现，含有C-末端一半的MoTA蛋白水解段(MoTA CTD)与DNA结合的表观解离常数与全长MoTA的解离常数相似。我们的结果支持一个模型，在该模型中，Mota NTD与sigma70的远C-末端区域以及Asia与sigma70之间的相互作用破坏了sigma70与DNA-35区域的接触，并促进了Mota CTD与-30 Mota盒基序的结合。

Normal cell development requires the regulation of transcription initiation and activation in order to express appropriate genes at appropriate times. We study regulation of transcription initiation using a simple prokaryotic system: E. coli RNA polymerase, a five subunit complex composed of a core (2 alphas, beta, and beta') and a sigma specificity factor. The sigma70 factor specifies transcription from promoters that are responsible for basal gene expression during vegetative growth. When sigma70 is present within polymerase, two of its domains, 2.4 (an internal region) and 4.2 (the C-terminal region), interact with sequences within the -10 and -35 regions, respectively, of promoter DNA. However, in free sigma70, DNA binding is prevented by domain 1.1, the N-terminal region of the protein. To investigate the effect of domain 1.1 on the DNA binding properties of sigma70 when it is present in polymerase, we used various promoters to compare polymerases with and without domain 1.1 in in vitro transcription assays and in assays assessing the formation and decay of stable, pretranscription complexes. We found that depending on the promoter, the absence of domain 1.1 can promote or inhibit transcription initiation by affecting the formation of stable pretranscription complexes. However, domain 1.1 does not affect the stability of these complexes once they are formed. For polymerase containing domain 1.1, the efficiency of stable complex formation correlates with how well the -10 and -35 regions of a promoter match the ideal sigma70 recognition sequences. However, when domain 1.1 is absent, having this match becomes less important in determining how efficiently stable complexes are made. Our work is the first to demonstrate that domain 1.1 has a regulatory role when sigma70 is present in polymerase. We suggest that domain 1.1 influences initiation by constraining polymerase to assess a promoter primarily by the fitness of its -10 and -35 regions to the canonical sequences. The bacteriophage T4 proteins MotA, a transcriptional activator, and AsiA, a co-activator, interact with the sigma70 subunit of RNA polymerase and switch its promoter specificity from host promoters to T4 middle promoters. Middle promoters contain the sigma70 recognition sequences at -10 but lack the canonical -35 sequences. Instead they contain a 9 bp motif (a MotA box) which is centered at -30. MotA binds the MotA box as well as interacts with sigma70. We have found that the N-terminal half of MotA (MotA NTD), which is thought to include the activation domain, interacts with the C-terminal region of sigma70 in an E. coli 2-hybrid assay. Either replacement of the C-terminal 17 residues of sigma70 with comparable residues from another sigma, sigma38, or the sigma70 mutation R608C, located near the end of sigma70's 613 amino acids, is defective in this assay. These results suggest that the far C-terminal region of sigma70 is important for the MotA/sigma70 interaction. We have also found that a proteolyzed fragment of MotA that contains the C-terminal half (MotA CTD) binds DNA with an apparent dissociation constant that is similar to that of full length MotA. Our results support a model in which the interaction between MotA NTD and the far C-terminal region of sigma70 and between AsiA and sigma70 serve to disrupt sigma70 contacts with the -35 region of the DNA and to facilitate the binding of MotA CTD to the -30 MotA box motif.