Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
J Mol Biol. 2014 Feb 6;426(3):550-7. doi: 10.1016/j.jmb.2013.11.001. Epub 2013 Nov 8.
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.
由与 GFP（绿色荧光蛋白）（或其他荧光蛋白）融合的 LacI 和 TetR 阻遏蛋白可视化的染色质整合的 lacO 和 tetO 操纵子位点阵列被广泛用于监测各种系统中染色体基因座的行为。然而，这种阵列的插入和相应蛋白质的表达已知会破坏基因组结构。在几种情况下，推测位于不同染色体上的此类阵列的并置反映了相应基因座的配对。在这里，我们报告了一种突变 GFP 二聚化以破坏 GFP 二聚化的 TetR-GFP 版本（TetR-A206KGFP 或“TetR-kGFP”），体内消除了 tetO 阵列的配对，并使带有这些阵列的染色体基因座的空间接近度恢复到野生型水平。这些数据表明，阵列的配对是由 GFP 二聚化引起的，因此提供了蛋白质辅助染色体相互作用的一个例子。用另一种蛋白质 TetR-tdTomato 标记的阵列，其倾向于形成分子内二聚体而不是分子间二聚体，也显示出配对水平降低，支持这一观点。TetR-kGFP 为研究具有低配对背景的染色体基因座提供了一个改进的系统。
