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.
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