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, P1 and P2, 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 several critical discoveries made last year. The major mode of regulation of gal transcription is achieved by formation of a DNA-multiprotein complex, called repressosome, which causes repression of both promoters. Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping. Previous experiments suggested that the DNA loop contains 113 bp encompassing the promoter region. Interaction between two DNA-bound proteins would be allowed if the binding sites on DNA are properly aligned. To test the idea that the observed repression of gal transcription in vitro is mediated by DNA looping, we investigated the effect of changing the relative angular orientation of the two GalR binding sites, OE and OI in the DNA helix. We found that repression is a periodic function of the distance between the two operator sites. Since repression recurred commensurate with DNA helical repeat, we concluded that the observed in vitro repression is mediated by DNA looping and the in vitro conditions reflect the in vivo situation. Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure. Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding. Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA. We also investigated the dependency of HU binding to DNA on GalR binding to the two operators (cooperativity). We showed that GalR piggybacks HU to the critical position on the DNA through a specific GalR-HU interaction. This is the first example of HU making a specific contact with another protein. The GalR-HU contact resulting in cooperative binding of the two proteins to DNA may be transient and absent in the final repressosome structure. A sequence- independent DNA-binding protein being recruited to an architectural site on DNA through a specific association with a regulatory protein may be a common mode for assembly of complex nucleoprotein structures.
| 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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