Bacteriophage T4 Gene Expression

在大肠杆菌感染过程中，噬菌体T4篡夺宿主的转录机制，将其重定向到早期、中期和晚期噬菌体基因的表达。这台机器是由E.大肠杆菌RNA聚合酶，与所有细菌聚合酶一样，由具有RNA合成活性的亚基（β、β ′、α 1、α 2和ω）和特异性因子（σ）的核心组成。 sigma蛋白通过识别并结合启动子DNA内的序列元件来识别转录的起始。在指数增长期间，E.大肠杆菌是sigma70，它像所有初级sigma一样，由四个区域组成。Sigma70分别使用其中心部分（区域2和3）和C末端部分（区域4）中的残基识别宿主启动子DNA的位置-10和-35周围的DNA元件。此外，区域4内的残基还必须与核心聚合酶内的结构（称为β-瓣）相互作用，以定位σ 70区域4，使其能够接触-35 DNA。 T4接管E。大肠杆菌RNA聚合酶通过噬菌体编码的因子的作用与聚合酶相互作用，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个元件，分别被sigma70区域2和4识别。然而，尽管T4中间启动子与σ 70 - 10元件具有极好的匹配，但是它们具有以σ 70 - 35元件为中心的噬菌体元件（MotA盒）而不是σ 70 - 35元件。需要两种T4编码的蛋白质，DNA结合激活剂（MotA）和T4编码的共激活剂（AsiA）来激活中间启动子。AsiA单独抑制一大类大肠杆菌的转录。大肠杆菌启动子通过结合和结构重塑σ 70区域4，防止其与-35元件和β-瓣的相互作用。除了其抑制活性，AsiA诱导的重塑提出了一个表面可访问MotA结合sigma70区4在一个过程中称为西格玛拨款。 虽然MotA的N-末端（NTD）和C-末端（CTD）的结构是可用的，但不存在全长MotA或MotA的任何部分与DNA的结构。我们通过将MotA内的单个特异性半胱氨酸与切割试剂溴乙酰氨基苄基-EDTA（FeBABE）缀合并确定用MotA/AsiA/RNA聚合酶/DNA复合物获得的FeBABE切割位点来产生MotA及其DNA元件的分子图。 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的C末端和NTD/CTD接头与MotA盒元件的5'部分对齐。 使用表面等离子体共振，我们表明，上游和下游的MotA盒序列的序列是需要最佳的结合和MotA单独非常迅速地从DNA解离。 我们的研究结果提出了一个模型，其中MotA快速采样的DNA，只有稳定结合，一旦它也可以从事其蛋白质的合作伙伴，AsiA重塑sigma70存在于RNA聚合酶。

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.