Bacteriophage T4 Gene Expression

为了实现其发育周期，T4噬菌体接管其宿主大肠杆菌的RNA聚合酶。与所有细菌聚合酶一样，大肠杆菌RNA聚合酶由具有RNA合成活性的核心亚基（β、β′、α 1、α 2和ω）和特异性因子（sigma）组成，特异性因子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，大肠杆菌的初级sigma为sigma70。Sigma70识别宿主启动子DNA -10和-35位置附近的DNA元件，分别使用其中心部分（区域2和3）和c端部分（区域4）的残基。此外，区域4内的残基还必须与核心聚合酶内的结构相互作用，称为β -flap，以定位sigma70区域4，使其能够接触-35 DNA。T4通过噬菌体编码因子的作用接管大肠杆菌RNA聚合酶，这些因子与聚合酶相互作用并改变其对启动子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sigma70 -10元件非常匹配，但它们有一个以-30为中心的噬菌体元件（MotA box），而不是sigma70 -35元件。激活中间启动子需要两个t4编码蛋白，一个dna结合激活因子（MotA）和一个t4编码的辅激活因子（AsiA）。单独的AsiA通过结合并在结构上重塑sigma70区域4来抑制大肠杆菌启动子的转录，阻止其与-35元件和β -皮瓣的相互作用。除了其抑制活性外，asia诱导的重塑被认为可以使MotA在一个称为sigma占用的过程中与sigma70区域4结合。

To achieve its developmental cycle, bacteriophage T4 takes over the RNA polymerase of its host, E. coli. E. coli RNA polymerase, like all bacterial polymerases, is composed of a core of subunits (beta, beta', alpha1, alpha2, and omega), which have RNA synthesizing activity, and a specificity factor (sigma), which 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. 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. MotA is a two domain protein that that has been shown to interact with both region 4 of sigma70 and a promoter element, the MotA box. In a collaboration with the laboratory of Dr. Milton Werner (Rockefeller University), we have defined the face of MotA that recognizes sigma70 region 4. NMR chemical shift analysis indicates that MotA uses a basic/hydrophobic cleft to interact with the C-terminus of AsiA-remodeled sigma70, but MotA does not interact with AsiA itself. Mutations within this cleft, at residues K3, K28, and Q76, both impair the interaction of MotA with sigma70 region 4 and MotA-dependent activation of transcription. Furthermore, mutations at these residues greatly decrease phage viability. Most previously described activators that target sigma70 directly use acidic residues to engage a different, basic surface of region 4. Our more recent work is now defining the molecular surface of the far C-terminal region of sigma70 that interacts with MotA and with the beta-flap. Our results suggest that this patch of sigma70 uses similar residues to interact with either the activator or the beta domain. Our work supports accumulated evidence indicating that sigma appropriation by MotA and AsiA uses a fundamentally different mechanism to activate transcription. Besides the MotA/AsiA-dependent activation of T4 middle promoters, middle RNA is also produced by the extension of early transcription into middle genes, which are positioned downstream of early gene(s) and an early promoter. Thus, this RNA is time-delayed since it cannot be synthesized until the elongating RNAP reaches the downstream middle genes. There is indirect evidence to suggest that that a T4 anti-termination process may be involved in this extension. However, there is still no direct evidence to support the existence of such a system. Because T4 RNA is produced by two pathways, a T4 motA- phage is still able to grow, albeit poorly, in a wild type E. coli host. However, work in another lab has demonstrated that a T4 motA mutation is lethal in the E.coli strain tabG. We have now shown that the mutations within tabG that are responsible for the T4 motA- growth defect are within rpoB, the beta subunit of RNA polymerase. Though the tabG rpoB mutations are separated by over 1200 base pairs in the DNA sequence, the two mutations are close in the structures of RNA polymerase that have been reported for thermophilic bacteria. One mutation is located near the wall of the RNA exit channel. The other mutation is immediately adjacent to a hydrophobic pocket, which is thought to be responsible for the separation of the RNA from the DNA-RNA hybrid. Our primer extension analysis demonstrates that in a T4 wild type infection of E. coli containing the tabG mutations, strain B11, less middle RNA is generated from T4 middle promoters, and in a T4 motA- infection of strain B11, there is much less RNA made overall. Our preliminary results suggest that the surfaces of beta identified by the rpoB mutations may be involved in MotA/AsiA activation of middle promoters and/or in the synthesis of middle RNA through the extension of early RNAs into middle genes. Understanding how these mutations affect both processes will yield new insights into transcription initiation and elongation.