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. 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. 2. Intra-chromosomal connections. 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. 3. 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. 4. DNA looping. 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. 4. Single DNA molecule studies: The lambda DNA loop, its different geometric forms and their stability were determined by tethered particle motion (TPM) analysis.
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