Regulation of Gene Transcription

类核结构：从我们之前的遗传研究中，我们提出大肠杆菌类核具有确定的结构，这决定了它的转录谱。这是基于我们的发现：类核蛋白（HU）的突变显著地改变了转录模式。为了遵循这个想法，我们想知道类核结构。我们知道(i) HU参与染色体折叠；（ii）长期以来人们都知道有一种或多种未知RNA参与染色体的形成；（iii）我们也知道HU与RNA结合。我们测定了HU在大肠杆菌中的RNA结合谱。我们设计了一种Rip-chip实验来鉴定与HU结合的RNA种类。它们是：80个trna，所有rRNA， 5个非编码rna和29个mRNA片段。其中一种非编码RNA （non5）与染色体上数百个DNA序列同源。非5 DNA序列（有一个或两个不匹配）分布在染色体周围，该序列在染色体的每个位点上经常以重复单位出现。我们提出，一个hu -non - 5 RNA复合物结合到每个non - 5 DNA位点上，然后聚集形成一个多结构域的类核结构（以前通过电子显微镜在染色体上看到）。每个结构域具有不同的超螺旋，影响其组成启动子的转录。为了测试HU-RNA介导的DNA结构域的形成，我们目前正在构建一个9KB的质粒DNA，其中有四个非5 DNA位点位于不同的标记位置。根据我们的模型，我们建议在存在HU和非5 RNA的情况下，在AFM下观察质粒，以观察假定的三叶草叶结构。GalR的类核折叠：通过使用3C（染色体构象捕获）测定，我们已经证明GalR作为一种调节子特异性转录因子靶向大肠杆菌染色体周围的数百个结合位点。我们已经证明GalR与这些位点结合并在dna结合时结合，以帮助形成染色体中的三级结构。染色体的这种特殊折叠有助于DNA在类核中凝聚。我们认为其他类核蛋白（HNS， HU等）通过类似的机制帮助染色体凝聚。类核结构和转录：我们之前已经证明，在大肠杆菌中的基因表达模式与野生型的类核蛋白突变体HU发生了巨大的变化。我们通过DNA平铺阵列实验发现，这种变化起源于转录水平。这些结果与HU参与染色体折叠的模型一致；一种折叠会产生一种转录谱，另一种折叠会产生不同的转录谱。腺苷酸环化酶CRP相互作用：与cAMP结合的CRP蛋白激活大肠杆菌中许多基因的转录。cAMP是由腺苷酸环化酶从ATP合成的。虽然只有100微摩尔cAMP存在于细胞中，但在野生型细胞中，它负责最大数量的cAMP- crp介导的转录，但在腺苷酸环化酶缺陷突变细胞中，需要添加510 mM cAMP才能进行高水平转录。cAMP对细胞没有渗透性屏障。我们通过表明腺苷酸环化酶与CRP结合并通过隧道直接传递产物（cAMP）而不扩散到介质中，解决了这个悖论。这使得cAMP可以在低得多的cAMP浓度下激活转录。我们通过(i)两种蛋白相互交联的能力，以及（ii）分离和表征CRP的非接触突变体来证明环化酶CRP的接触。用腺苷酸环化酶和ATP代替cAMP，在体外偶联转录系统中再现了高效率的转录。DNA环在噬菌体lambda转录调控中：我们和其他人之前已经证明了DNA环在噬菌体状态下对噬菌体lambda基因的调控中存在。噬菌体蛋白CI可抑制两个裂解基因启动子PL和PR的转录，这两个启动子相距3kb。CI通过绑定它们的同源算子元素OL1、OL2、OL3和OR1、OR2、OR3来起作用。CI通过结合OR2、OR3和OL3位点调节自身合成。这种调节需要通过连接两组由操作符结合的CI分子介导的操作符来形成DNA环。我们已经通过使用不同的操作突变体以不同的组合在纯化系统中证实了DNA环的调节。单DNA分子研究：用系留粒子运动（TPM）分析确定了lambda DNA环、其不同的几何形状及其稳定性。

Nucleoid Structure: From our previous genetic study, we proposed that E. coli nucleoid has a defined structure, which dictates its transcription profile. This was based on our finding that a mutation in the nucleoid protein, HU, altered the transcription pattern dramatically. To follow this idea, we wanted to know the nucleoid structure. We knew that (i) HU participates chromosome folding; (ii) It was known for a long time that one or more unknown RNA participates in chromosome formation; (iii) It was also known that HU binds to RNA. We determined the RNA binding profile of HU in E. coli. We devised a Rip-chip assay to identify the RNA species that bind to HU. They are: 80 tRNAs, all rRNA, 5 non-coding RNAs, and segments of 29 mRNA. One of the non-coding RNA (non5) is homologues to hundreds of DNA sequences in the chromosome. The non5 DNA sequences (with one or two mismatch) are distributed around the chromosome, and the sequence is present frequently in repeating units at each locus in the chromosome. We proposed that an HU-non5 RNA complex binds to each of the non5 DNA loci and then aggregated to give rise to a multi domain nucleoid structure (previously seen in the chromosome by Electron Microscope). Each domain has different superhelecity, which influences the transcription of its constituent promoters. To test the HU-RNA mediated DNA domain formation, we are currently constructing a 9KB plasmid DNA, in which there are four non5 DNA sites placed at different marked positions. We propose to look at the plasmid under AFM in the presence of HU and non5 RNA to see, according to our model, a putative clover leaf structure. Nucleoid folding by GalR: By the use of 3C (chromosome conformation capture) assays, we have shown that GalR, identified as a regulon specific transcription factor targets several hundred binding sites around the E. coli chromosome. We have shown that GalR bind to these sites and associate while DNA-bound to help formation of a tertiary structure in the chromosome. This kind of specific folding of the chromosome helps DNA condensation in the nucleoid. We believe other nucleoid proteins (HNS, HU, etc) help chromosome condensation by similar mechanisms. Nucleoid structure and transcription: We have previously shown that gene expression patterns in E. coli dramatically changes from the wild type pattern in mutants of the nucleoid protein, HU. We have found by DNA tiling array experiments that such changes originate at the level of transcription. These results are consistent with the model that HU participates in folding chromosome; one kind of fold gives one kind of transcription profile, and another kind of fold a different profile. Adenylate cyclase CRP interaction: CRP protein which binds to cAMP, activates transcription of numerous genes in E. coli. cAMP is made by the enzyme adenylate cyclase from ATP. Although only 100 micro molar cAMP, present in the cell, it is responsible for maximal amount of cAMP-CRP mediated transcription in the wild type cell, 510 mM cAMP needs to be added to an adenylate cyclase defective mutant cell for high level transcription. There is no permeability barrier to cAMP to cell. We resolved the paradox by showing that adenylate cyclase binds to CRP and deliver the product (cAMP) directly by tunneling without diffusion to the medium. This makes it possible for cAMP to activate transcription at a much lower cAMP concentration. We demonstrated the cyclase CRP contact by (i) cross-linking ability of the two proteins to each other, and (ii) isolating and characterizing non-contacting mutants of CRP. The high efficiency of transcription was re-produced in an in vitro coupled transcription system by replacing cAMP by adenylate cyclase and ATP. DNA looping in phage lambda transcription regulation: We and others have previously shown the existence of DNA loops in the regulation of phage lambda genes in a prophage state. The phage protein CI represses transcription from its two promoters for lytic gene transcription, PL and PR, located 3 KB apart. CI acts by binding to their cognate operator elements, OL1, OL2, OL3 and OR1, OR2, OR3 respectively. CI regulates its own synthesis by binding to OR2, OR3 and OL3 loci. This regulation requires formation of a DNA loop by linking two sets of operators mediated by the operators-bound CI molecules. We have confirmed the regulations by DNA looping in a purified system by the use of different operator mutants in various combinations. Single DNA molecule studies: The lambda DNA loop, its different geometric forms and their stability were determined by tethered particle motion (TPM) analysis.