通过将光遗传学控制与显微镜相结合来探究启动子如何在酿酒酵母中解码 TF 动力学的方案。

Protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy.

机构信息

Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI 53705, USA.

出版信息

STAR Protoc. 2024 Sep 20;5(3):103002. doi: 10.1016/j.xpro.2024.103002. Epub 2024 Jul 13.

DOI:10.1016/j.xpro.2024.103002

PMID:39003745

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11284393/

Abstract

Gene promoters filter transcription factor (TF) localization dynamics and changes in the DNA binding affinity of TFs. Here, we present a protocol to probe how promoters decode TF dynamics in Saccharomyces cerevisiae by combining optogenetic control with microscopy. We describe steps for preparing and characterizing a light delivery platform and light-controlled TF mutants. We then detail procedures for subjecting the TFs to light doses that generate defined patterns of localization while measuring fluorescent reporter gene activation via live-cell microscopy. For complete details on the use and execution of this protocol, please refer to Sweeney and McClean..

摘要

基因启动子过滤转录因子（TF）的定位动态和 TF 的 DNA 结合亲和力的变化。在这里，我们通过将光遗传学控制与显微镜结合，提出了一种探测启动子如何在酿酒酵母中解码 TF 动态的方案。我们描述了制备和表征光传递平台和光控 TF 突变体的步骤。然后，我们详细介绍了使 TF 接受光剂量的程序，这些光剂量可以在通过活细胞显微镜测量荧光报告基因激活的同时产生明确定义的定位模式。有关此方案的使用和执行的完整详细信息，请参阅 Sweeney 和 McClean。