RNA polymerase II transcription initiation complex

Project Summary. The goal of this project is to define the interactions between RNA polymerase II, the basal
transcription factors, and the chromatin template that lead to accurate transcription initiation and productive
elongation. Using the many approaches available in the yeast Saccharomyces cerevisiae model system,
fundamental aspects of gene expression will be studied. Specific Aim 1 will continue our studies of pre-
initiation complex (PIC) assembly using colocalization single-molecule TIRF microscopy. The interaction
dynamics of multiple basal transcription factors on the transcription template are imaged in real time using
nuclear extracts, which provide a more physiological context than purified systems. Our experiments to date
have already revealed unexpected transient intermediates and branched pathways not visible in ensemble
assays, necessitating revisions to current models for PIC assembly. These studies will be expanded to look at
additional factors, as well as to how promoter sequences and nucleosomes affect factor dynamics. Specific
Aim 2 will focus on how TATA-binding protein (TBP) and TBP-associated factors (TAFs), which together make
up the basal factor TFIID, interact to recognize promoters to nucleate PIC formation. Both single molecule
colocalization and FRET experiments will test recent models suggesting TFIID conformation changes occur
upon DNA binding. Specific Aim 3 will continue our studies of co-transcriptional histone modifications. Our
immobilized template in vitro transcription system has been extended to chromatinized templates, and we find
it can reproduce multiple histone modifications linked to transcription. The dynamics of the relevant modifying
enzymes will be tested by quantitative mass spectrometry and single molecule colocalization experiments to
determine if they remain associated with elongation complexes or get transferred to nucleosomes in passing.
The effect of pre-existing histone modifications will also be probed. For all three aims, interesting findings will
be validated in vivo using the genetic, genomic, and molecular techniques our lab has developed over many
years. Although this project uses a model system, mechanisms of transcription are highly conserved in
eukaryotes and, based on past experience, the results will almost certainly be directly applicable to human
gene expression. This fundamental knowledge is essential for understanding how mutations in transcription
factors and histone modifying enzymes lead to diseases such as cancer and developmental defects.

Grant Number: 5R01GM046498-33
NIH Institute/Center: NIH
Principal Investigator: Stephen Buratowski