ABSTRACT
Numerous biological processes are regulated by DNA elements that communicate with their targets over a distance via formation of protein-bridged DNA loops. One of the first questions arising in studies of DNA looping is whether the rate of loop formation is limited by diffusion of the DNA sites. We addressed this question by comparing the in vitro measured rates of transcription initiation in the NtrC-glnAp2 enhancer-dependent transcription initiation system with predictions of two different theoretical models. The promoter and enhancer were in a 7.6-kb plasmid and separated by 2.5 kb. The measurements were performed for different values of the plasmid superhelix density, from 0 to -0.07. Earlier theoretical analysis, based on the Monte Carlo simulation of DNA conformations, showed that if the rate of loop formation is determined by the equilibrium probability of juxtaposition of the DNA sites, the rate should be approximately 100 times higher in supercoiled than in relaxed DNA. On the other hand, Brownian dynamics simulation showed that if the rate of loop formation is limited by the site diffusion, it should be nearly independent of DNA supercoiling. We found that efficiency of the transcription initiation increases by nearly two orders of magnitude as a result of the corresponding increase of the template supercoiling. This clearly shows that the rate of bridging in the enhancer-promoter system is not limited by diffusion of the DNA sites to one another. We argue that this conclusion derived for the specific system is likely to be valid for the great majority of biological processes involving protein-mediated DNA looping.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/ultrastructure , DNA/chemistry , DNA/ultrastructure , Models, Chemical , Models, Molecular , Binding Sites , Computer Simulation , Models, Statistical , Nucleic Acid Conformation , Protein Binding , Protein ConformationABSTRACT
Insulators are DNA sequences that are likely to be involved in formation of chromatin domains, functional units of gene expression in eukaryotes. Insulators can form domain boundaries and block inappropriate action of regulatory elements (such as transcriptional enhancers) in eukaryotic nuclei. Using an in vitro system supporting enhancer action over a large distance, the enhancer-blocking insulator activity has been recapitulated in a highly purified system. The insulator-like element was constructed using a sequence-specific DNA-binding protein making stable DNA loops (lac repressor). The insulation was entirely dependent on formation of a DNA loop that topologically isolates the enhancer from the promoter. This rationally designed, inducible insulator-like element recapitulates many key properties of eukaryotic insulators observed in vivo. The data suggest novel mechanisms of enhancer and insulator action.
Subject(s)
Enhancer Elements, Genetic/genetics , Base Sequence , Chromatin/genetics , Cloning, Molecular , DNA, Superhelical/chemistry , DNA, Superhelical/genetics , Drug Design , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Promoter Regions, Genetic , Restriction Mapping , Templates, Genetic , Transcription, GeneticABSTRACT
Enhancers are regulatory DNA sequences that can work over a large distance. Efficient enhancer action over a distance clearly requires special mechanisms for facilitating communication between the enhancer and its target. While the chromatin looping model can explain the majority of the observations, some recent experimental findings suggest that a chromatin scanning mechanism is used to establish the loop. These new findings help to understand the mechanism of action of the elements that can prevent enhancer-promoter communication (insulators).
