We have been studying the mechanism by which initiation of transcription is regulated by activators and repressors, by using the galactase operon in Escherichia coli as a model system. The gal operon is transcribed by two tanden promoters, P 1 and P 2, which are regulated in a variety of ways by a number of regulatory proteins, which act by binding to their corresponding sites on the gal DNA. We report two critical finding below. (1) Transcription from P 1 is activated by cAMP and its receptor protein (CRP) complex which when bound to DNA enhances gal transcription. We have set up an assay using the fluorophore, 2,aminopurine to directly analyze the isomerization step of initiation. We have been able to show that isomerization can be further subdivided into three steps, one of which is rate limiting. We have also shown that cAMP-CPR stimulate P 1 transcription by enhancing the rate limiting step of isomerization. (2) Both promoters of the gal operon are repressed by the formation of a DNA loop. Loop formation requires the interaction of two GalR regulators, when bound to two operators, which span the promoters, in the presence of the histone-like protein HU. HU binds to a site between the two operators. By structure-based genetic analysis, we have defined the GalR interfaces for interaction. The interaction generates a V-shaped structure of stacked GalR. We are currently studying the role of HU in DNA-looping by isolating and characterizing HU mutants which are defective in DNA looping by GalR but bind to DNA.

National Institute of Health (NIH)
Division of Basic Sciences - NCI (NCI)
Intramural Research (Z01)
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Basic Sciences
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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
Zwieb, Christian; Adhya, Sankar (2009) Plasmid vectors for the analysis of protein-induced DNA bending. Methods Mol Biol 543:547-62
Lia, Giuseppe; Semsey, Szabolcs; Lewis, Dale E A et al. (2008) The antiparallel loops in gal DNA. Nucleic Acids Res 36:4204-10
Adhya, Sankar L; Court, Donald L; Friedman, David I et al. (2008) Amos Oppenheim (31 October 1934 to 24 September 2006). Mol Microbiol 67:685-6
Lewis, Dale E A; Komissarova, Natalia; Le, Phuoc et al. (2008) DNA sequences in gal operon override transcription elongation blocks. J Mol Biol 382:843-58
Rokney, Assaf; Kobiler, Oren; Amir, Amnon et al. (2008) Host responses influence on the induction of lambda prophage. Mol Microbiol 68:29-36
Edgar, Rotem; Rokney, Assaf; Feeney, Morgan et al. (2008) Bacteriophage infection is targeted to cellular poles. Mol Microbiol 68:1107-16
Court, Donald L; Oppenheim, Amos B; Adhya, Sankar L (2007) A new look at bacteriophage lambda genetic networks. J Bacteriol 189:298-304
Guo, Fusheng; Adhya, Sankar (2007) Spiral structure of Escherichia coli HUalphabeta provides foundation for DNA supercoiling. Proc Natl Acad Sci U S A 104:4309-14
Semsey, Szabolcs; Virnik, Konstantin; Adhya, Sankar (2006) Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA. J Mol Biol 358:355-63

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