Bacteriophage T4 Gene Expression

为了完成其发育周期，噬菌体T4接管了其宿主大肠杆菌的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合成活性的亚基(β、β‘、α1、α2和omega)和特异性因子(Sigma)组成，后者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，大肠杆菌的主要西格玛是西格玛70。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。 MoTA是一种两个结构域的蛋白质，已被证明与Sigma70的第4区和启动子元件MoTA盒相互作用。在与洛克菲勒大学Milton Werner博士的实验室合作下，我们定义了MOTA的表面，识别sigma70区域4。核磁共振化学位移分析表明MOTA使用基本/疏水裂隙与亚洲重塑的sigma70的C末端相互作用，但MOTA不与亚洲本身相互作用。该裂隙内K3、K28和Q76残基的突变都破坏了MoTA与sigma70区域4的相互作用和MoTA依赖的转录激活。此外，这些残基的突变极大地降低了噬菌体的生存能力。大多数先前描述的靶向sigma70的激活剂直接使用酸性残基与区域4的不同碱性表面结合。我们最近的工作是定义sigma70的远C-末端区域的分子表面，该区域与MoTA和β-Flat相互作用。我们的结果表明，sigma70的这个补丁使用类似的残基与激活剂或贝塔结构域相互作用。我们的工作支持了积累的证据，即Mota和Asia使用根本不同的机制来激活转录。 除了依赖于MoTA/Asia的T4中间启动子的激活外，中间基因也是通过早期转录延伸到中间基因而产生的，中间基因位于早期基因(S)和早期启动子的下游。因此，这种RNA是时间延迟的，因为它不能被合成，直到延长的RNAP到达下游的中间基因。有间接证据表明，T4反终止过程可能参与了这一延长。然而，目前仍没有直接证据支持这种制度的存在。 因为T4RNA是由两条途径产生的，所以T4噬菌体仍然能够在野生型大肠杆菌宿主中生长，尽管很差。然而，另一家实验室的研究表明，T4MoTA突变在大肠杆菌菌株TabG中是致命的。我们现在已经证明，导致T4 Mota-Growth缺陷的TabG内的突变位于rpoB内，rpoB是RNA聚合酶的Beta亚单位。尽管TabG rpoB突变在DNA序列中相差超过1200个碱基对，但这两个突变在RNA聚合酶的结构中非常接近，这两个突变已被报道用于嗜热菌。一个突变位于RNA出口通道的壁上。另一个突变紧挨着一个疏水口袋，人们认为这个口袋负责将RNA从DNA-RNA杂交物中分离出来。我们的引物延伸分析表明，在含有tabG突变的T4野生型大肠杆菌B11株中，T4中间启动子产生的中间RNA较少，而在T4 Mota感染的B11株中，合成的总RNA要少得多。我们的初步结果表明，rpoB突变所确定的β表面可能参与了中间启动子的MoTA/Asia激活和/或通过将早期RNA延伸到中间基因来合成中间RNA。了解这些突变是如何影响这两个过程的，将对转录启动和延伸产生新的见解。

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.