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Research has been conducted on the Hox family of proteins. The identification and characterization of the regions important for the transcription activation by the Drosophila Hox protein Ultrabithorax carried out via the use of deletion and point mutants in the regions distant from the homeodomain are discussed.

Results demonstrate the significance of the hinge region of lactose repressor in high affinity DNA binding, sequence selectivity, and in the allosteric response. Temperature studies indicate that disulfide in the repressor alters the protein-DNA interaction

Research has been conducted on purine repressor (PurR) that binds to specific DNA and lactose repressor (LacI) binding. The differences between these proteins are discussed.

Operator DNA sequence variation has been found to enhance high-affinity binding by hinge helix mutants of lactose-repressor protein. The mechanism used by genetic regulatory proteins to tell what the specific DNA sequences are was explored in some detail. Binding of a LacI mutant series to other variant operator DNA sequences showed the importance of symmetry-related bases, spacing and the central base pair sequence in high-affinity complex formation.

A study was conducted to analyze four single-tryptophan mutants of lac repressor protein from Escherichia coli. Phosphorescence and optically detected magnetic resonance measurements were carried out. Mutations in the lac repressor gene were produced in the pAC1 plasmid. Results indicated that the influence of inducer binding correlate with a conformation change that alters specific polar interactions occurring at each affected tryptophan site.

Lactose repressor protein structural transitions were examined with regard to unfolding and dissociation in the presence of varying concentrations of urea which were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and functional activities. A single cooperative transition with no evidence of folded dimeric or monomeric species of this protein was revealed by the spectroscopic data.

The regions of the lac repressor protein involved in conformational changes in response to ligand binding in Escherichia coli were determined. Mutant repressor proteins with single tryptophans were produced by mutating each of the two native tryptophan residues to tyrosine and altering the residue of interest to tryptophan. Findings showed that in the investigated areas of the lac repressor, the inducer-bound form is different from the conformation of the unliganded form.

The result of water release on the ultrabithorax homeodomains (Ubx-HD) and deformed proteins homeodomains (Dfd-HD) DNA binding affinity differences was evaluated through measurements of sensitivity of protein-DNA binding constant to osmotic pressures generated by neutral solutes. Results have shown that the binding of the DNA with Ubx-HD can be increased by enhancing the osmolality of the bulk solution. Dfd-HD, however, does not exhibit the same sensitivity to osmotic conditions.

Asp(super 274) present at the bottom of the binding site in the lac repressor protein is necessary for the binding of the protein to sugar. The conformational changes in the protein due to sugar binding increase the binding affinity. Protein molecules whose Asp(super 274) has been replaced by Asn or Glu binds sugar less than that the wild-type protein. The conformational changes in the Asn substituted protein are similar to that of the wild-type protein while that of the Glu substituted protein are opposite.

Linkage between protein-protein, protein-DNA and protein-ligand binding is essential for the molecular mechanism of transcriptional regulation that is responsive to cellular and extracellular environmental signals. The property of two different dimeric variants of the lactose repressor reveals that the criteria for intersubunit interaction affect the functional propensity of the oligomeric form for the target site of DNA-and the regulatory function in the cell.

An analysis of lac repressor subunit interfaces reveals that coiled-coil structure formation is an important component of tetramer formation in the lac repressor. A stable long-axis dimer formation confirms the presence of two separate interfaces in tetrameric repressor assembly. Site-specific insertion mutagenesis adds leucine heptal repeat units to the C-terminus.

Phosphorescence and optical defection of magnetic resonance (ODMR) studies on the E. coli lactose repressor (Lac Rep) and the two single point mutants W220Y and W201Y are used to analyze the low temperature triplet state properties of two Trp residues and their reactions to monosaccharide IPTG and disaccharide melibiose inducer sugars. Native Lac Rep's phosphorescence spectrum identifies the Trp201 residue in a buried 0, 0 band polarized region at 413.7 nanometers in W220Y. The partially exposed Trp220 residue has a broad blue shifted 0,0 band at 409. 7 nanometers in W201Y. Trp220 has a 0, 0 band and 0.8 nanometer red shift in W220Y for both IPTG and melibiose inducers whereas Trp201 binds more to ITPG than to melibiose.

DNA loop formation plays an important role in the regulation of DNA metabolism. These cellular processes include transcriptional initiation in the operons of prokaryotic organisms and inhibition of phage systems. In eukaryotic systems, DNA looping was found in steroid hormone receptor proteins and homeodomain proteins. DNA-looped structures were also detected in recombination and replication in both the eukaryotic and prokaryotic cells. DNA loop formation is influenced by the distance between the binding sites, DNA sequence and other proteins which affect conformational behavior in genetic sequencing.

Abstract Although yeast two-hybrid experiments are commonly used to identify protein interactions, the frequent occurrence of false negatives and false positives hampers data interpretation. Using both yeast one-hybrid and two-hybrid experiments, we have identified potential sources of these problems: the media preparation protocol and the source of the yeast nitrogen base may not only impact signal range but also effect whether a result appears positive or negative. While altering media preparation may optimize signal differences for individual experiments, media preparation must be reported in detail to replicate studies and accurately compare results from different experiments.

Acidic, basic, polar and apolar mutations are introduced at positions 94-98 in order to explore that a disproportionate number of substitutions in the regions of the monomer-monomer interface has altered the complex features of the Lacl allosteric response. The results have confirmed the critical role of amino acids 94-98 and have shown that the N-subdomain interface has formed a primary pathway in Lacl allosteric response.

Targeted molecular dynamics (TMD) techniques were employed to simulate the transition between inducer- and operator-bound states and understand allosteric mechanism related to lactose repressor protein (Lacl)-mediated transcription in Escherichia coli. The double mutant D149C/S193C purified from cell extracts showed decreased sensitivity to inducer binding while retaining wild-type binding affinities and kinetic constants for both operator and inducer.

Research describes biochemical characterization of three of the LacI mutationsin the core pivot. Simulated targeted molecular dynamics analysis indicate that he mutations alter the behavior of LacI protein in tems of repressor function.

Linking changes in amino acid sequences to the evolution of transcription regulatory domains is often complicated by the low sequence complexity and high mutation rates of intrinsically disordered protein regions. For the Hox transcription factor Ultrabithorax (Ubx), conserved motifs distributed throughout the protein sequence enable direct comparison of specific protein regions, despite variations in the length and composition of the intervening sequences. In cell culture, the strength of transcription activation by Drosophila melanogaster Ubx correlates with the presence of a predicted helix within its activation domain. Curiously, this helix is not preserved in species more divergent than flies, suggesting the nature of transcription activation may have evolved. To determine whether this helix contributes to Drosophila Ubx function in vivo, wild-type and mutant proteins were ectopically expressed in the developing wing and the phenotypes evaluated. Helix mutations alter Drosophila Ubx activity in the developing wing, demonstrating its functional importance in vivo. The locations of activation domains in Ubx orthologues were identified by testing the ability of truncation mutants to activate transcription in yeast one-hybrid assays. In Ubx orthologues representing 540 million years of evolution, the ability to activate transcription varies substantially. The sequence and the location of the activation domains also differ. Consequently, analogous regions of Ubx orthologues change function over time, and may activate transcription in one species, but have no activity, or even inhibit transcription activation in another species. Unlike homeodomain-DNA binding, the nature of transcription activation by Ubx has substantially evolved. [ABSTRACT FROM AUTHOR]