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，像所有细菌聚合酶一样，它由具有RNA合成活性的亚基(β、β‘、α1、α2和omega)的核心和一个特异性因子(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。除了其抑制活性外，亚洲诱导的重塑还允许MoTA的N末端结构域(Mota NTD)与sigma70的C末端结合，以及Mota的C末端结构域(Mota CTD)与Mota box结合。这个过程被称为西格玛挪用。 尽管有几十种激活剂晶体结构和RNA聚合酶结构，但激活剂/RNA聚合酶/DNA复合体只有一个完整的结构。然而，这种结晶复合体执行的激活类型与Sigma专有的激活类型有根本的不同。我们以前将生化分析、可用结构和建模结合在一起，开发了西格玛拨款的结构模型。我们的工作描述了ASIA/MOTA如何将sigma70重定向到T4中间启动子DNA，从而使RNA聚合酶活性重定向到T4，以及sigma70区域4的灵活性对这一过程可能是如何至关重要的。我们的工作表明，MOTA通过一种先前未知的相互作用机制与其DNA结合基序相互作用，在这种相互作用机制中，CTD的双翼螺旋结构与DNA的主槽接触，接头与次槽接触。与Steve White(St Judes)博士的实验室合作，我们用DNA解决了MoTA连接物-CTD的晶体结构，揭示了一种新的蛋白质-DNA相互作用模式。CTD结构域主要通过与DNA骨架的相互作用与DNA结合，但在特定的同源结构中，通过与主槽和次槽中的MoTA盒基序的额外相互作用，结合被增强。连接两个MoTA结构域的连接子在通过微小沟槽相互作用稳定复合体方面起着关键作用。该结构与我们之前的模型一致，该模型是通过使用整个转录复合体进行化学切割实验得出的。α-和β-D-葡萄糖基-5-羟甲基脱氧胞嘧啶取代了T4DNA中的胞嘧啶，对接模拟表明同源结构中的一个空腔可以容纳修饰后的胞嘧啶。我们的结合研究证实，修饰显著增强了MoTA与DNA的结合亲和力。我们的工作揭示了DNA修饰如何扩展小DNA基序的唯一性，以促进蛋白质-DNA相互作用的特异性。

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 has 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 those 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, the AsiA-induced remodeling allows the N-terminal domain of MotA (MotA NTD) to bind to the C-terminus of sigma70 and the C-terminal domain of MotA (MotA CTD) to bind to the MotA box. This process is called sigma appropriation. Despite dozens of activator crystallographic structures and RNA polymerase structures, there is only one complete structure of an activator/RNA polymerase/DNA complex. However, the type of activation performed by this crystallized complex is fundamentally different from that of sigma appropriation. We previously combined biochemical analyses, available structures, and modeling to develop a structural model of sigma appropriation. Our work depicted how AsiA/MotA redirects sigma70, and therefore RNA polymerase activity, to a T4 middle promoter DNA and how the flexibility of sigma70 region 4 is likely crucial for this process. Our work suggested that MotA interacts with its DNA binding motif using a previously unidentified interaction mechanism in which the double wing helix structure of the CTD contacts the major groove of the DNA and the linker contacts the minor groove. In collaboration with the laboratory of Dr. Steve White (St Judes), we solved the crystal structure of the MotA linker-CTD with the DNA, revealing a new mode of protein-DNA interaction. The CTD domain binds DNA mostly via interactions with the DNA backbone, but the binding is enhanced in the specific cognate structure by additional interactions with the MotA box motif in both the major and minor grooves. The linker connecting the two MotA domains plays a key role in stabilizing the complex via minor groove interactions. The structure is consistent with our previous model derived from chemical cleavage experiments using the entire transcription complex. Alpha- and beta-D-glucosyl-5-hydroxymethyl-deoxycytosine replace cytosine in T4 DNA, and docking simulations indicate that a cavity in the cognate structure can accommodate the modified cytosine. Our binding studies have confirmed that the modification significantly enhances the binding affinity of MotA for the DNA. Our work reveals how a DNA modification can extend the uniqueness of small DNA motifs to facilitate the specificity of protein-DNA interactions.