The long-term goal of this project is to understand how transcription by RNA polymerase II (RNApII) is coupled to RNA processing and termination. Earlier funding periods of this project 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 the """"""""pausing"""""""" of RNApII at developmentally regulated genes in embryonic stem cells. 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 current funding period of the parent grant, four specific aims are being pursued. The first is to understand the two known termination pathways (for polyA and non-polyA transcripts) and how RNApII chooses between them.
A second aim studies the biogenesis of short unstable transcripts produced by divergent transcription at many RNApII promoters.
The third aim uses NET-Seq to probe the role of CTD phosphorylation in RNA elongation, pausing, and termination.
The fourth aim exploits our discovery that the Rpb1 CTD can be transferred onto a different RNApII subunit, asking whether the different CTD modifications and functions need to occur in """"""""cis"""""""" on the same CTD or can be split between two CTDs. This supplement will add one new specific aim attempting to answer whether there are functional differences between CTD repeats at different positions. In the first subaim, a CTD will be engineered that adds several lysines and non-consensus repeats so that mass spectrometry can be used to distinguish proximal and distal repeats. The in vivo phosphorylations on these CTD fragments will be analyzed in wild-type cells and mutants of various CTD modifying enzymes. In the second subaim, a series of CTD mutants will be constructed that incorporate a non-native, photoreactive amino acid in vivo. In vivo crosslinking and analysis of associated proteins will show whether CTD-associated factors preferentially associate with proximal or distal repeats. Together, the results of these experiments will show whether the CTD phosphorylation patterns and associated proteins are distinct or identical in different regions of the CTD.
Improper gene expression causes many diseases, including developmental defects and cancer. The goal of this project is to understand the fundamental processes by which genes are expressed and regulated. This understanding will be essential for designing treatments and drugs to restore normal gene expression in diseased cells or to alter gene expression patterns to create pluripotent stem cells and specific differentiated cell types.
|Suh, Hyunsuk; Ficarro, Scott B; Kang, Un-Beom et al. (2016) Direct Analysis of Phosphorylation Sites on the Rpb1 C-Terminal Domain of RNA Polymerase II. Mol Cell 61:297-304|
|Soares, Luis M; Radman-Livaja, Marta; Lin, Sherry G et al. (2014) Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns. Cell Rep 6:961-72|
|Marquardt, Sebastian; Escalante-Chong, Renan; Pho, Nam et al. (2014) A chromatin-based mechanism for limiting divergent noncoding transcription. Cell 157:1712-23|
|Heo, Dong-hyuk; Yoo, Inhea; Kong, Jiwon et al. (2013) The RNA polymerase II C-terminal domain-interacting domain of yeast Nrd1 contributes to the choice of termination pathway and couples to RNA processing by the nuclear exosome. J Biol Chem 288:36676-90|
|Suh, Hyunsuk; Hazelbaker, Dane Z; Soares, Luis M et al. (2013) The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits. Mol Cell 51:850-8|
|Hazelbaker, Dane Z; Marquardt, Sebastian; Wlotzka, Wiebke et al. (2013) Kinetic competition between RNA Polymerase II and Sen1-dependent transcription termination. Mol Cell 49:55-66|
|Soares, Luis M; Buratowski, Stephen (2013) Histone Crosstalk: H2Bub and H3K4 Methylation. Mol Cell 49:1019-20|
|Fowler, Trent; Suh, Hyunsuk; Buratowski, Stephen et al. (2013) Regulation of primary response genes in B cells. J Biol Chem 288:14906-16|
|Soares, Luis M; Buratowski, Stephen (2012) Yeast Swd2 is essential because of antagonism between Set1 histone methyltransferase complex and APT (associated with Pta1) termination factor. J Biol Chem 287:15219-31|
|Hazelbaker, Dane Z; Buratowski, Stephen (2012) Transcription: base J blocks the way. Curr Biol 22:R960-2|
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