Bacteriophage T4 Gene Expression

正常的细胞发育需要调控转录起始和激活，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研究转录起始的调控：大肠杆菌RNA聚合酶，一个由核心（2 α， β和β '）和sigma特异性因子组成的五个亚基复合物。sigma70因子指定在营养生长过程中负责基础基因表达的启动子的转录。当sigma70存在于聚合酶中时，它的两个结构域2.4（一个内部区域）和4.2 （c端区域）分别与启动子DNA -10和-35区域内的序列相互作用。然而，在游离sigma70中，DNA结合被结构域1.1（蛋白质的n端区域）阻止。为了研究1.1结构域对聚合酶中sigma70的DNA结合特性的影响，我们在体外转录实验中使用了不同的启动子来比较有1.1结构域和没有1.1结构域的聚合酶，以及评估稳定的转录前复合物的形成和衰变的实验。我们发现，根据启动子的不同，域1.1的缺失可以通过影响稳定的转录前复合物的形成来促进或抑制转录起始。然而，结构域1.1不影响这些配合物一旦形成的稳定性。对于含有1.1结构域的聚合酶，稳定复合物形成的效率与启动子的-10和-35区域与理想的sigma70识别序列的匹配程度有关。然而，当结构域1.1缺失时，在确定稳定复合物的制备效率方面，这种匹配就变得不那么重要了。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通过限制聚合酶来评估启动子，主要是通过其-10和-35区域对标准序列的适应度来影响起始。噬菌体T4蛋白MotA（转录激活因子）和AsiA（共激活因子）与RNA聚合酶sigma70亚基相互作用，将其启动子特异性从宿主启动子转换为T4中间启动子。中间启动子包含-10的sigma70识别序列，但缺乏规范的-35序列。相反，它们包含一个以-30为中心的9 bp motif （MotA box）。MotA绑定MotA盒子并与sigma70相互作用。我们发现，在大肠杆菌2-杂交实验中，MotA的n端一半（MotA NTD）被认为包括活化结构域，与sigma70的c端区域相互作用。将sigma70的c -末端17个残基替换为另一个sigma， sigma38或sigma70突变R608C（位于sigma70的613个氨基酸末端），在本实验中都是有缺陷的。这些结果表明sigma70的远c端区域对MotA/sigma70相互作用很重要。我们还发现含有c端一半的MotA蛋白水解片段（MotA CTD）与DNA结合，其明显的解离常数与全长MotA相似。我们的研究结果支持一个模型，即MotA NTD与sigma70的远c端区域之间以及AsiA与sigma70之间的相互作用有助于破坏sigma70与DNA -35区域的接触，并促进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.