Nucleoid Structure: Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli. HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro. We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known. Role of GalR in nucleoid structure: By microscopic analysis of fluorescent-labeled GalR, a regulon-specific transcription factor in Escherichia coli, we observed that GalR is present in the cell as aggregates (one to three fluorescent foci per cell) in nongrowing cells. To investigate whether these foci represent GalR-mediated association of some of the GalR specific DNA binding sites (gal operators), we used the chromosome conformation capture (3C) method in vivo. Our 3C data demonstrate that, in stationary phase cells, many of the operators distributed around the chromosome are interacted. By the use of atomic force microscopy, we showed that the observed remote chromosomal interconnections occur by direct interactions between DNA-bound GalR not involving any other factors. Mini plasmid DNA circles with three or five operators positioned at defined loci showed GalR-dependent loops of expected sizes of the intervening DNA segments. Our findings provide unique evidence that a transcription factor participates in organizing the chromosome in a three-dimensional structure. We believe that these chromosomal connections increase local concentration of GalR for coordinating the regulation of widely separated target genes, and organize the chromosome structure in space, thereby likely contributing to chromosome compaction. DNA looping in Bacteriophage Lambda DNA in gene regulation. The prophage state of bacteriophage wavelength is extremely stable and is maintained by a highly regulated level of wavelength repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P(RM). CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P(RM) regulation. We additionally report that different configurations of DNA loops are central to this regulation--one configuration further enhances autoactivation and another is essential for autorepression of P(RM).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC008751-33
Application #
8763018
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
33
Fiscal Year
2013
Total Cost
$1,257,590
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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Semsey, Szabolcs; Krishna, Sandeep; Erdossy, Janos et al. (2009) Dominant negative autoregulation limits steady-state repression levels in gene networks. J Bacteriol 191:4487-91
Zurla, Chiara; Manzo, Carlo; Dunlap, David et al. (2009) Direct demonstration and quantification of long-range DNA looping by the lambda bacteriophage repressor. Nucleic Acids Res 37:2789-95
Zwieb, Christian; Adhya, Sankar (2009) Plasmid vectors for the analysis of protein-induced DNA bending. Methods Mol Biol 543:547-62
Lee, Sang Jun; Lewis, Dale E A; Adhya, Sankar (2008) Induction of the galactose enzymes in Escherichia coli is independent of the C-1-hydroxyl optical configuration of the inducer D-galactose. J Bacteriol 190:7932-8