Research Projects

Combinatorial Code of CTD

The conformational states of the C-terminal domain (CTD) of eukaryotic RNA polymerase II represent a critical regulatory check point for transcription. The CTD, found only in eukaryotes, consists of 26-52 tandem hepta-peptide repeats with the general consensus sequence,1TyrSerProThrSerProSer7. The CTD can spatially and temporally recruit different regulatory and processing factors to the transcriptional machinery (Fig.1). CTD regulates the transcription through its various conformations, which are achieved through post-translational covalent modifications or prolyl isomerization (Fig. 1). Phosphorylation of serine residues at positions 2 and 5 is the primary modification sites whose patterns have been correlated to various stages of transcription.

(1) The phosphatase families of CTD Ser5 phosphatases.

The phosphorylation states of CTD, namely the “CTD code“ are coordinated by CTD kinases and phosphatases. CTD phosphatases are especially difficult to study because of the high heterogeneity in endogenous CTD phosphorylation pattern. The removal of phosphorylation labels in a precise and timely manner is equally essential as placing the label for the interpretation of the CTD code during transcription regulation. For example, Ssu72 and Fcp have been reported to play key roles in general transcription, mRNA processing/termination and RNA polymerase II recycling. In contrast to those general regulators, human Scp only affects transcription of specific neuronal genes. We solved the structure of Scp and Ssu72 phosphatases which recognize the same CTD sequence but with dramatically different transcription outcome. We are further investigating the association of these phosphatases with binding partners from the transcription complexes they are involved with and how such interaction play a major role for their biological function in transcription regulation.

(2) The cross-talk of Ser5 phosphorylation and Pro6 isomerization

CTD regulation of transcription is mediated both by phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. We provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but that have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation.

(3) Chemical compounds for neural regeneration.

Scp proteins are phosphatases that target phosphorylated Ser5 (phos.Ser5) in the hepta-repeats of CTD. Identified as a modulator of neural gene silencing, Scp proteins act as evolutionarily conserved transcriptional co-repressors; and in this role, they can inhibit neuronal gene transcription in non-neuronal cells. We identified the first selective inhibitor for Scp protein, which is also the first reported selectivity inhibitor for HAD family. We are currently developing this scaffold for compounds that can promote neuron regeneration. Such compounds are not only useful as a tool to study the cascade of neuronal gene expression pattern, more importantly, it has the potential as a chemical agent promoting new neuron growth which will greatly benefit patients in neurodegenerative diseases such as Alzheimer’s.