Understanding the Mechanisms of Human RNA Polymerase II Transcription using Single Molecule Fluorescence Colocalization

Abstract

Transcription is the first step in cellular gene expression and a key regulatory point for determining how a cell responds to stimuli. In mammalian cells, transcription is initiated by the formation of a pre-initiation complex (PIC), an assembly of general transcription factors (GTFs) and RNA polymerase II (Pol II), on promoter DNA. Our goal is to use single molecule approaches to address mechanisms of PIC formation and activity that have previously been unattainable using traditional biochemical approaches. We have developed a novel single molecule total internal reflection fluorescence (TIRF) microscopy assay that allows us to visualize human Pol II transcriptional activity on individual DNA templates. With this single molecule setup, we have performed experiments to address the pathways by which active pre-initiation complexes can form. Ultimately we found that Pol II and three GTFs can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA.We are also developing a single molecule system to monitor dynamic protein-protein interactions that facilitate activation of transcription. It has long been known that transcriptional activators interact with subunits of transcription factor IID (TFIID) to potentiate high levels of transcription, however, the biophysical parameters defining these interactions are almost entirely uncharacterized. We have developed a novel method to label transcriptional activators and monitor their interactions with transcription factor TFIID in real-time using single molecule fluorescence colocalization. We observe that the activation domains from different activators exhibit different kinetic parameters for interactions with TFIID. We are positioned to address previously unanswered questions regarding how important protein-protein interactions between TFIID and transcriptional activators impact gene expression.