Biomedical subjects

Konstantin Virnik

Publications and source records attributed to Konstantin Virnik.

4 recordsLinked to original sources

PubMed2006

Three-stage regulation of the amphibolic gal operon: from repressosome to GalR-free DNA.

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.

Binding Sites↗

PubMed2005

A gamut of loops: meandering DNA.

Nucleoprotein complexes comprising short DNA loops (150 base pairs or less) are involved in a wide variety of DNA transactions (e.g. transcription regulation, replication and recombination) in both prokaryotes and eukaryotes, and also can be useful in designing nanostructures. In these higher-order nucleoprotein complexes, proteins bound to spatially separated sites on a DNA interact with each other by looping out the relatively stiff intervening DNA. Recent technological developments have enabled determination of DNA trajectories in a few DNA-loop-containing regulatory complexes. Results show that, in a given system, a specific DNA trajectory is preferred over others.

Bacterial Proteins↗

PubMed2004

DNA trajectory in the Gal repressosome.

The Gal repressosome is a higher-order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli. During the repressosome assembly, a DNA loop is formed by the interaction of two GalR dimers, bound to two spatially separated operators, OE and OI, flanking the gal promoters. Structure-based genetic analysis indicated that GalR homodimers interact directly and form a V-shaped stacked tetramer in repressosome, further stabilized by HU binding to an architecturally critical position on the DNA. In this scheme of GalR tetramerization, the alignment of the operators in the DNA loop could be in either parallel (PL) or antiparallel (AL) mode. As each mode can have two alternative geometries differing in the mutual stacking of the OE- and OI-bound GalR dimers, it is possible to have four different DNA trajectories in the repressosome. Feasibilities of these trajectories were tested by in vitro transcription repression assays, first by isolating GalR mutants with altered operator specificity and then by constructing four different potential loops with mutant GalR heterodimers bound to specifically designed hybrid operators in such a way as to give rise to only one of the four putative trajectories. Results show that OE and OI adopt a mutual antiparallel orientation in an under-twisted DNA loop, consistent with the energetically optimal structural model. In this structure the center of the HU-binding site is located at the apex of the DNA loop. The approach reported here can be used to distinguish between otherwise indistinguishable DNA trajectories in complex nucleoprotein machines.

Bacterial Proteins↗

PubMed2003

"Antiparallel" DNA loop in gal repressosome visualized by atomic force microscopy.

DNA looping is often involved in positive and negative regulation of gene transcription in both prokaryotes and eukaryotes. The transcription of the gal operon of Escherichia coli from two overlapping promoters P1 and P2 is negatively regulated via Gal repressosome assembly. It involves binding of two dimeric Gal repressor proteins (GalR) to two operators, O(E) and O(I), flanking the two promoters, and formation of 113 bp DNA loop due to tetramerization of the two bound GalR dimers. The process requires negatively supercoiled DNA and the presence of the histone-like protein HU. Previous modeling of the repressosome based on evaluation of DNA elastic energy suggested a mutual antiparallel, rather than parallel, orientation of the two gal operators in an under-twisted DNA loop. To visualize the Gal loop by atomic force microscopy (AFM), plasmid DNA molecules were constructed with increased distance between the two operators. The AFM results demonstrated the formation of an antiparallel DNA loop in the Gal repressosome consistent with our earlier hypothesis. Importantly, the overall shape of the GalR mediated loop proved to be indistinguishable from that in the chimerical loop of the same size containing two lac operators (instead of two gal operators) and formed by LacI. In addition, a possibility of the gal operon repression mediated by GalR in the absence of HU was shown in the new DNA constructs. Implications of these findings for the DNA structural organization in bacterial nucleoid are discussed.

Bacterial Proteins↗