ABSTRACT The C-terminal domain of RNA polymerase II (CTD) is a unique structure in eukaryotes that coordinates various transcriptional factors to Pol II through its post-translational modifications. The simple consensus sequence of CTD (Y1S2P3T4S5P6S7) is repeated many times consecutively and is mostly conserved. The phosphorylation of the two SP motifs in the sequence has been identified to be essential for every round of transcription but the other three residues, Y1, T4 and S7, also have functional implications during transcription. To understand the molecular mechanism of these post-translational modifications of CTD on transcription modulation, we combine a novel mass spectrometry methodology with biochemical and biophysical studies to investigate how the identity and modification states of these three positions affect the post-translational modification states of RNA polymerase II. We will then examine the consequences of altering the modification patterns of CTD on transcription, which will allow us to understand this fundamental mechanism that regulates transcription.