Search

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.

During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties. Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain, Full-length Ultrabithorax bound to target DNA 2.5-fold weaker than its isolated homeodomain. Using N-terminal and C-terminal deletion mutants, we demonstrate that the YPWM region and the disordered microexons (termed the 11 region) inhibit DNA binding ~2-fold, whereas the disordered 12 region inhibits homeodomain-DNA interaction a further ~40-fold. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal 174 amino acids (R region) in a length-dependent manner. Both the 12 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. Given that (i) the Ii region and a portion of the R region alter homeodomain-DNA binding as a function of pH and (ii) an internal deletion within Ii increases Ultrabithorax-DNA affinity, Ii must directly impact homeodomain-DNA interaction energetics. However, 12 appears to indirectly affect DNA binding in a manner countered by the N terminus. The amino acid sequences of 12 and much of the Ii and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions. [ABSTRACT FROM AUTHOR]

The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain β-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; π-stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74rsquo;. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data. [ABSTRACT FROM AUTHOR]

The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development. Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown. Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0-12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein. The Ubx·DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis. Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction. Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1. A large region of Ubx, including the repression domain, is required for interaction with DIP1. These more variable sequences may be key to the differential Hox function observed in vivo. The Ubx.DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx. The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA. The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated. [ABSTRACT FROM AUTHOR]

The crystal structures of lactose repressor protein (LacI) provide static endpoint views of the allosteric transition between DNA- and IPTG-bound states. To obtain an atom-by-atom description of the pathway between these two conformations, motions were simulated with targeted molecular dynamics (TMD). Strikingly, this homodimer exhibited asymmetric dynamics. All asymmetries observed in this simulation are reproducible and can begin on either of the two monomers. Asymmetry in the simulation originates around D149 and was traced back to the pre-TMD equilibrations of both conformations. In particular, hydrogen bonds between D149 and S193 adopt a variety of configurations during repetitions of this process. Changes in this region propagate through the structure via noncovalent interactions of three interconnected pathways. The changes of pathway 1 occur first on one monomer. Alterations move from the inducer-binding pocket, through the N-subdomain beta-sheet, to a hydrophobic cluster at the top of this region and then to the same cluster on the second monomer. These motions result in changes at (1) side chains that form an interface with the DNA-binding domains and (2) K84 and K84', which participate in the monomer-monomer interface. Pathway 2 reflects consequent reorganization across this subunit interface, most notably formation of a H74-H74rsquo; pi-stacking intermediate. Pathway 3 extends from the rear of the inducer-binding pocket, across a hydrogen-bond network at the bottom of the pocket, and transverses the monomer-monomer interface via changes in H74 and H74rsquo;. In general, intermediates detected in this study are not apparent in the crystal structures. Observations from the simulations are in good agreement with biochemical data and provide a spatial and sequential framework for interpreting existing genetic data.