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。我们正试图用几种方法来理解这种结构。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 （nc5）与分布在染色体周围的数百个DNA序列同源（有一个或两个不匹配）；该序列经常以重复单位出现在染色体的每个位点上。我们提出ncRNA-HU复合体有助于大肠杆菌染色体结构的形成。我们现在已经证明，与上述建议一致，与野生型细胞不同，HU基因、NC5基因缺失的细胞，或HU和NC5基因都缺失的细胞，在电镜下表现为染色体去致密化。Intra-chromosomal连接。GalR的类核折叠：通过使用3C（染色体构象捕获）测定，去年我们发现GalR，被确定为一种调控特异性转录因子，靶向大肠杆菌染色体周围的数百个结合位点。我们证明GalR与这些位点结合，而dna结合有助于染色体三级结构的形成。染色体的这种特殊折叠有助于DNA在类核中凝聚。在过去的一年里，我们通过另外两种方法证实了这种结构：(i)用荧光蛋白标记的GalR在每个细胞中显示出大约2-3个荧光点，表明GalR与DNA位点结合形成复合物。（II）在原子力显微镜下，一个GalR结合位点分别形成了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。我们正在研究噬菌体λ抑制蛋白（CI）的多水平自调节，通过体外DNA环和嵌入式遗传网络，利用对操作位点的不对称识别来平衡噬菌体生活方式的溶原和裂解模式。今年，我们已经证明，破坏CI与相邻操作符0R1和0R2的合作结合，会导致CI与替代操作符对0R2和0R3的合作结合，从而导致CI合成的关闭，从而导致裂解生长。gal操纵子。今年，我们研究了在DNA环介导的启动子抑制条件下，RNA聚合酶与galP1启动子-35区域的具体接触。这项工作已经发表。目前，我们正在准备一份关于单个碱基对在穿插gal启动子转录起始中的作用的手稿。

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.