Regulation of Transcription The gal operon of Escherichia coli is negatively regulated by the Gal repressosome, a higher order nucleoprotein complex containing a DNA loop that encompasses two gal promoters. In the repressosome structure, Gal repressor (GalR) dimers are bound to the two operator sites, flanking the promoter region, thus generating a DNA loop. The DNA loop is stabilized by binding of the architectural HU protein to the apex of the loop, and negative supercoiling. The gal promoters are also regulated in opposite directions by GalR without DNA looping. The repressosome-mediated as well as looping-independent transcription regulation of the two promoters is lifted in the presence of the inducer D-galactose. We tested the effect of D-galactose on various DNA-protein and protein-protein interactions of different regulatory complexes and on transcription repression in vitro . We found that the inducer breaks up the repressosome with clear intermediates in a concentration-dependent manner. The sequential disassembly generates different stages of regulation of the gal operon. The D-galactose-dependent switch from one stage of regulation to another satisfies the amphibolic requirement of the gal operon. Interactions between proteins bound to distant sites along a DNA molecule require bending and twisting deformations in the intervening DNA. In certain systems, the sterically allowed protein-DNA and protein-protein interactions are hypothesized to produce loops with distinct geometries that may also be thermodynamically and biologically distinct. For example, theoretical models of Gal repressor/HU-mediated DNA-looping suggest that the antiparallel DNA loops, A1 and A2, are thermodynamically quite different. They are also biologically different, since in experiments using DNA molecules engineered to forms only one of the two loops, the A2 loop failed to repress in vitro transcription. Surprisingly, single molecule measurements show that both loop trajectories form and that they appear to be quite similar energetically and kinetically. Many of the gal regulon genes are activated in the presence of the adenosine cyclic-3,5-monophosphate (cAMP)- cAMP receptor protein (CRP) complex. We studies transcriptional regulation of the gal regulon promoters simultaneously in a purified system and attempted to integrate the two small molecule signals, D-galactose and cAMP, that modulate the isoregulators and CRP respectively, at each promoter, using Boolean logic. Results show that similarly organized promoters can have different input functions. We also found that in some cases the activity of the promoter and the cognate gene can be described by different logic gates. We combined the transcriptional network of the galactose regulon, obtained from our experiments, with literature data to construct an integrated map of the galactose network. Structural analysis of the network shows that at the interface of the genetic and metabolic network, feedback loops are by far the most common motif. Prokaryotic RNA polymerases are capable of efficient, continuous synthesis of RNA in vivo , yet purified polymerase-DNA model systems for RNA synthesis typically produce only a limited number of catalytic turnovers. Here, we report that the ribosomal protein S1--which plays critical roles in translation initiation and elongation in Escherichia coli and is believed to stabilize mRNA on the ribosome--is a potent activator of transcriptional cycling in vitro . Deletion of the two C-terminal RNA-binding modules--out of a total of six loosely homologous RNA-binding modules present in S1--resulted in a near-loss of the ability of S1 to enhance transcription, whereas disruption of the very last C-terminal RNA-binding module has only a mild effect. We propose that, in vivo , cooperative interaction of multiple RNA-binding modules in S1 may enhance the transcript release from RNA polymerase, alleviating its inhibitory effect and enabling the core enzyme for continuous reinitiation of transcription. Nucleoid Structure and Function We determined the crystal structure of the Escherichia coli nucleoid-associated HUalphabeta protein by x-ray diffraction and observed that the heterodimers form multimers with octameric units in three potential arrangements, which may serve specialized roles in different DNA transaction reactions. It is of special importance that one of the structures forms spiral filaments with left-handed rotations. A negatively superhelical DNA can be modeled to wrap around this left-handed HUalphabeta multimer. Whereas the wild-type HU generated negative DNA supercoiling in vitro , an engineered heterodimer with an altered amino acid residue critical for the formation of the left-handed spiral protein in the crystal was defective in the process, thus providing the structural explanation for the classical property of HU to restain negative supercoils in DNA. In bacteria, the contribution of global nucleoid organization in determining cellular transcription programs is unclear. Using a mutant form of the most abundant nucleoid-associated protein HU, HUalpha (E38K, V42L), we previously showed that nucleoid remodeling by the mutant protein re-organizes the global transcription pattern. Here, we demonstrate that, unlike the dimeric wild-type HU, HUalpha (E38K,V42L) is an octamer and wraps DNA around its surface. The formation of wrapped nucleoprotein complexes by HUalpha (E38K,V42L) leads to a high degree of DNA condensation. The DNA wrapping is right-handed, which restrains positive supercoils. In vivo , HUalpha (E38K,V42L) shows altered association and distribution patterns with the genetic loci who transcription are differentially affected in the mutant strain.
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| 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 |
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| 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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