Abstract
Chromosomally integrated arrays of lacO and tetO operator sites visualized by LacI and TetR repressor proteins fused with GFP (green fluorescent protein) (or other fluorescent proteins) are widely used to monitor the behavior of chromosomal loci in various systems. However, insertion of such arrays and expression of the corresponding proteins is known to perturb genomic architecture. In several cases, juxtaposition of such arrays located on different chromosomes has been inferred to reflect pairing of the corresponding loci. Here, we report that a version of TetR-GFP mutated to disrupt GFP dimerization (TetR-A206KGFP or “TetR-kGFP”) abolishes pairing of tetO arrays in vivo and brings spatial proximity of chromosomal loci marked with those arrays back to the wild-type level. These data argue that pairing of arrays is caused by GFP dimerization and thus presents an example of protein-assisted interaction in chromosomes. Arrays marked with another protein, TetR-tdTomato, which has a propensity to form intramolecular dimers instead of intermolecular dimers, also display reduced level of pairing, supporting this idea. TetR-kGFP provides an improved system for studying chromosomal loci with a low pairing background.
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