Bacteriophage T4 Gene Expression

在感染大肠杆菌的过程中，T4噬菌体侵占了宿主的转录机制，使其重定向到表达早、中、晚噬菌体基因。这一机制是由大肠杆菌RNA聚合酶驱动的，与所有细菌聚合酶一样，该聚合酶由具有RNA合成活性的亚基(β、β‘、α1、α2和omega)和特异性因子(Sigma)组成。Sigma蛋白通过识别和结合启动子DNA中的序列元件来识别转录的开始。在指数生长过程中，大肠杆菌的主要Sigma是sigma70，它像所有的主要Sigma一样，由四个区域组成。Sigma70识别宿主启动子DNA-10和-35位置周围的DNA元件，分别使用其中心部分(区域2和3)和C末端部分(区域4)的残基。此外，区域4中的残基还必须与核心聚合酶中的结构相互作用，称为β-翻盖，以定位sigma70区域4，使其能够接触-35 DNA。 T4通过噬菌体编码的因子与聚合酶相互作用而接管大肠杆菌RNA聚合酶，并改变其对启动子DNA的特异性。早期T4启动子具有与宿主相似的-10和-35元件，分别由sigma70区2和4识别。然而，尽管T4中间启动子与sigma70-10元件有很好的匹配，但它们有一个以-30为中心的噬菌体元件(Mota盒)，而不是sigma70-35元件。激活中间启动子需要两个T4编码的蛋白质，一个是DNA结合激活子(MOTA)，一个是T4编码的共激活子(ASIA)。仅ASIA一项就通过结合和结构重塑sigma70区域4来抑制一大类大肠杆菌启动子的转录，阻止其与-35元件和β-Flat的相互作用。除了它的抑制活性外，亚洲诱导的重塑被认为是为了使MoTA能够在一个称为sigma专有的过程中与sigma70区域4结合。 尽管MOTA的N端(NTD)和C端(CTD)的结构是可用的，但没有全长MOTA的结构或带有DNA的MOTA的任何部分的结构。我们通过将MoTA中单一的特定半胱氨酸与切割试剂FeBABE(FeBABE)连接，并确定MoTA/ASIA/RNA聚合酶/DNA复合体获得的FeBABE切割位点，生成了MoTA及其DNA元件的分子图谱。我们的工作揭示了一种新的激活剂/DNA相互作用。MotACTD位于MoTA盒序列的3‘部分内，而MotANTD和NTD/CTD连接子的C末端与MoTA盒元件的5’部分对齐。利用表面等离子体共振，我们证明了MoTA盒序列的上游和下游序列都是最佳结合所必需的，并且MoTA单独一人很快就从DNA上解离了。我们的结果提出了一个模型，在这个模型中，MoTA快速采样DNA，只有当它也能与其蛋白质伙伴--存在于RNA聚合酶中的亚洲重塑的Sigma70--接触时，它才能稳定地结合。

During infection of Escherichia coli, bacteriophage T4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. This machinery is driven by E. coli RNA polymerase, which, like all bacterial polymerases, is composed of a core of subunits (beta, beta', alpha1, alpha2, and omega) that have RNA synthesizing activity and a specificity factor (sigma). The sigma protein identifies the start of transcription by recognizing and binding to sequence elements within promoter DNA. During exponential growth, the primary sigma of E. coli is sigma70, which, like all primary sigmas, is composed of four regions. Sigma70 recognizes DNA elements around positions -10 and -35 of host promoter DNA, using residues in its central portion (regions 2 and 3) and C-terminal portion (region 4), respectively. In addition, residues within region 4 must also interact with a structure within core polymerase, called the beta-flap, to position sigma70 region 4 so it can contact the -35 DNA. T4 takes over E. coli RNA polymerase through the action of phage-encoded factors that interact with polymerase and change its specificity for promoter DNA. Early T4 promoters, which have -10 and -35 elements that are similar to that of the host, are recognized by sigma70 regions 2 and 4, respectively. However, although T4 middle promoters have an excellent match to the sigma70 -10 element, they have a phage element (a MotA box) centered at -30 rather than the sigma70 -35 element. Two T4-encoded proteins, a DNA-binding activator (MotA) and a T4-encoded co-activator (AsiA), are required to activate the middle promoters. AsiA alone inhibits transcription from a large class of E. coli promoters by binding to and structurally remodeling sigma70 region 4, preventing its interaction with the -35 element and with the beta-flap. In addition to its inhibitory activity, AsiA-induced remodeling is proposed to make a surface accessible for MotA to bind to sigma70 region 4 in a process called sigma appropriation. Although structures for the N-terminal (NTD) and C-terminal (CTD) of MotA are available, no structures exist for full length MotA or any portion of MotA with DNA. We generated a molecular map of MotA with its DNA element by conjugating single, specific cysteines within MotA with the cleaving reagent, iron bromoacetamidobenzyl-EDTA (FeBABE) and determining the FeBABE cut sites obtained with the MotA/AsiA/RNA polymerase/DNA complex. Our work reveals a novel activator/DNA interaction. The MotACTD locates within the 3' portion of the MotA box sequence, while the C-terminal end of the MotANTD and the NTD/CTD linker align with the 5' portion of the MotA box element. Using surface plasmon resonance, we show that sequences both upstream and downstream of the MotA box sequence are needed for optimal binding and that MotA alone very quickly dissociates from the DNA. Our results suggest a model whereby MotA rapidly samples the DNA and is only stably bound once it can also engage its protein partner, the AsiA-remodeled sigma70 present within RNA polymerase.