Regulation of Gene Transcription

类核结构：根据我们先前的遗传学研究，我们提出E.大肠杆菌类核具有确定的结构，这决定了其转录谱。 这是基于我们的发现，即类核蛋白HU的突变显著改变了转录模式。 为了遵循这个想法，我们想知道类核的结构。 我们正试图用几种方法来理解这个结构。1. RNA的作用(i)我们知道HU参与染色体折叠;（ii）长期以来，我们知道一个或多个未知RNA参与染色体形成;（iii）我们也知道HU与RNA结合。 我们测定了HU在E.杆菌 我们设计了一种Rip-chip测定法来鉴定与HU结合的RNA种类。 它们是：80个tRNA片段、rRNA片段、12个非编码RNA片段和11个mRNA片段。其中一个非编码RNA（nc 5）与分布在染色体周围的数百个DNA序列（有一个或两个错配）同源;该序列经常以重复单位存在于染色体的每个位点。 我们认为ncRNA-HU复合物有助于E. coli染色体结构。我们现在已经证明，与上述提议一致，与野生型细胞不同，HU基因、NC 5基因或HU和NC 5基因两者缺失的细胞通过电子显微镜显示出去致密的染色体。2.染色体内连接。GalR的类核折叠：去年，我们利用3C（染色体构象捕获）分析，发现GalR作为一种调节子特异性转录因子，靶向大肠杆菌周围的数百个结合位点。coli染色体。 我们证明，GalR绑定到这些网站相关联，而DNA结合，以帮助在染色体中的三级结构的形成。 染色体的这种特殊折叠有助于DNA在类核中凝聚。 在过去的一年中，我们已经证实了这一结构，通过两个以上的方法：（i）GalR标记的荧光蛋白显示约2-3个荧光点，每个细胞显示GalR结合到DNA位点缔合形成复合物。(II)通过原子力显微镜观察，GalR结合位点分别显示形成3-5个确定大小的DNA环。 这些研究已于今年发表。转录调控：噬菌体λ。 我们正在研究多层次的自动调节噬菌体λ阻遏蛋白，CI，在体外的DNA循环和嵌入式遗传网络，它利用不对称识别的操作位点，以平衡溶原和裂解模式的噬菌体的生活方式。 今年，我们已经证明，破坏Cl与相邻操纵子0 R1和0 R2的协同结合带来Cl与替代操纵子对0 R2和0 R3的协同结合，导致Cl合成的关闭，从而导致裂解生长。 今年，我们研究了在DNA环介导的启动子抑制条件下，通过RNA聚合酶对galP 1启动子-35区域的特异性接触。 这项工作已经出版。 目前，我们正在准备一份手稿的作用，个别碱基对的转录起始散布半乳糖启动子。

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 are attempting to understand the structure using several approaches. 1. Role of RNA. (i) We knew that 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 fragments of tRNAs, rRNA, twelve non-coding RNAs, and segments of eleven mRNAs.One of the non-coding RNA (nc5) is homologues to hundreds of DNA sequences (with one or two mismatch) are distributed around the chromosome; the sequence is present frequently in repeating units at each locus in the chromosome. We proposed that ncRNA-HU complex help formation of the E. coli chromosome structure. We have now demonstrated, consistent with the above proposal, that cells with deletion of the HU genes, NC5 gene, or both HU and NC5 genes, unlike wild type cells, show decondensed chromosome by Electron Microscopy. 2. Intra-chromosomal connections. Nucleoid folding by GalR: By the use of 3C (chromosome conformation capture) assays, last year we showed that GalR, identified as a regulon specific transcription factor targets several hundred binding sites around the E. coli chromosome. We demonstrated that GalR bound to these sites 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. In the past year we have confirmed this structure by two more methods: (i) GalR labeled with fluorescent proteins showed about 2-3 fluorescent spots per cell showing GalR bound to DNA sites associate to form complexes. (II) A GalR binding sites showed formation of 3-5 DNA loops, respectively, of defined sizes by Atomic Force Microscopy. These studies have been published this year.Transcription Regulation:Bacteriophage lambda. We are investigating multilevel auto-regulation of bacteriophage lambda repressor protein, CI, by DNA looping in vitro and the embedded genetic network, which utilizes the asymmetric recognition of the operator site to balance the lysogenic and lytic modes of phage lifestyles. This year we have demonstrated that breaking up the co-operative binding of the CI to adjacent operators 0R1 and 0R2 brings up co-operative binding of CI to the alternative operators pair, 0R2 and 0R3 leading to shut off of CI synthesis and thus lytic growth.The gal operon. This year we investigated the specific contacts of the -35 region of the galP1 promoter by RNA polymerase under conditions of DNA looping mediated repression of the promoter. This work is already published. Currently, we are preparing a manuscript on the role of individual base pair in transcription initiation of interspersed gal promoters.