mRNA Capping Enzyme

Project Summary. The long-term goal of this project is to understand how transcription by RNA
polymerase II (RNApII) is coupled to RNA processing, chromatin modifications, and termination. This project
previously produced a model in which the C-terminal domain (CTD) of the RNApII subunit Rpb1 displays
characteristic phosphorylation patterns at different stages of the transcription cycle to promote binding of the
appropriate factors for co-transcriptional RNA processing. The fundamental knowledge generated by this
project provides significant insight into how the CTD phosphorylation cycle affects medically important
processes such as the stimulation of HIV transcription by the viral Tat protein and "pausing" of RNApII at
developmentally regulated genes. This project is necessary to better understand both the enzymes that
mediate the changes in CTD phosphorylation (kinases, phosphatases, etc.) as well as the proteins that
recognize these patterns.
In the next funding period, three specific aims will be pursued, with a focus on measuring dynamics of
events during transcription. The first aim continues our work directly analyzing CTD phosphorylation sites by
mass spectrometry. A modified CTD (msCTD) was engineered to directly assign phosphorylation sites by
mass, and in the previous period we developed a vastly improved peptide chromatography and analysis
pipeline. This will be used to analyze phosphorylation in yeast cells or extracts where various CTD modifying
enzymes are inactivated or depleted, and to determine the specificity of CTD binding proteins or CTD
antibodies. We will also extend msCTD analysis to mammalian and plant cells. The second aim studies three
proteins, each related to CTD phosphorylation and transcription elongation, that possess long “linker” domains
that apparently function as flexible arms. Using multiple approaches, we will probe how Tfb3 connects the
Kin28/Cdk7 kinase module to the body of TFIIH, how the Ctr9 “trestle” functions within the PAF complex to
facilitate transcription through nucleosomes, and how the Abd1 cap methyltransferase functions in elongation.
The third aim continues our single-molecule microscopy analysis of transcription elongation. We can visualize
individual transcription events with up to three fluorescently-labeled transcription factors, providing second to
millisecond time resolution of binding kinetics. We will measure the stoichiometries, order of binding, and
cooperative interactions between multiple CTD binding and elongation complex factors. Altogether, this project
will make a unique contribution to our understanding of gene expression by providing a time-resolved picture of
events that complements inherently static techniques such as structural studies or genomics.

Grant Number: 5R01GM056663-26
NIH Institute/Center: NIH
Principal Investigator: Stephen Buratowski