Researcher(s)
- Connor Kosinski, Biochemistry, University of Delaware
Faculty Mentor(s)
- Neal Zondlo, Chemistry & Biochemistry, University of Delaware
- Catherine Leimkuhler Grimes, Chemistry & Biochemistry, University of Delaware
Abstract
RNA polymerase II (RNA Pol II) is a vital protein, facilitating DNA transcription in all eukaryotes from yeast to humans. The C-terminal domain of RNA Pol II (RNA Pol II-CTD) comprises multiple tandem heptad repeats with the consensus sequence Y1S2P3T4S5P6S7. Up to 52 repeats of this sequence are found within the protein.1 These repeats, rich in Thr/Ser/Pro and Ser/Pro motifs, are known phosphorylation sites, which play a role in protein function by facilitating initiation and termination of transcription. Phosphorylation at serine-5 by TFIIH initiates transcription and recruits enzymes for 5’ capping, while phosphorylation at serine-2 by P-TEFb promotes elongation, splicing, and polyadenylation.2 These modifications induce structural changes that modulate RNA Pol II activity, but the structure is unknown due to its intrinsic disorder. Ssu72 phosphatase, acting on RNA Pol II-CTD, dephosphorylates SP motifs only in the cis-amide conformation, crucial for transcription termination.3 Phosphatases Fcp1 and Ssu72 dephosphorylate serine-2 and serine-5 respectively, playing essential roles in this process.2 Fmoc-L-4,4-difluoroproline (Fmoc-Dfp-OH) acts as a 19F-NMR probe, simplifying the measurement of the equilibrium constant Ktrans/cis by isolating the cis and trans 19F signals from the overlapping 1H signals. This constant provides structural information regarding the relative populations of cis and trans Ser-Pro amide bonds within the peptide sequence. Probing this structural change in the TSP sequence as a function of phosphorylation could provide a means to assess how these effects modulate RNA pol II activity. This hypothesis was tested to study the conformational effects of phosphorylation preceding proline through the synthesis of multiple YSPTSDfpS sequences with defined phosphorylation patterns.