Department of Chemistry, Texas A&M University.
Department of Chemistry, Texas A&M University;
J Vis Exp. 2020 Dec 26(166). doi: 10.3791/62113.
Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNA) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNA deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.
乙酰化组蛋白蛋白可在编码突变体 N-乙酰-赖氨酸（AcK）特异性甲烷八叠球菌吡咯赖氨酸 tRNA 合成酶（MmAcKRS1）及其同源 tRNA（tRNA）的大肠杆菌中轻易表达，从而组装具有特定乙酰化组蛋白的重组单核小体。MmAcKRS1 和 tRNA 在大肠杆菌中的翻译过程中，将 AcK 递送到所选 mRNA 的琥珀突变位点。该技术已广泛用于在 H3 赖氨酸位点掺入 AcK。吡咯赖氨酸 tRNA 合成酶（PylRS）也可以很容易地进化为掺入其他非典型氨基酸（ncAAs），用于蛋白质的特异性修饰或功能化。在这里，我们详细描述了一种使用 MmAcKRS1 系统将 AcK 掺入组蛋白 H3 并将乙酰化 H3 蛋白整合到重组单核小体中的方法。乙酰化的重组单核小体可用于生化和结合测定、结构测定等。获得修饰的单核小体对于设计与发现新相互作用和理解表观遗传学相关的实验至关重要。
