Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates

[Abstract] Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.

to productive elongation. These dynamic post-translational modifications and precisely recruited protein factors constitute the 'CTD Code' for eukaryotic transcription and ensure the production of mature and functional transcripts (Jeronimo et al., 2013;Mayfield et al., 2016).
RNAPII is recruited to the pre-initiation complex when the CTD is in the unphosphorylated form. Upon transcription initiation, the CTD becomes hyperphosphorylated, indicative of a transcriptionally engaged RNAPII. At transcription termination, the CTD undergoes dephosphorylation and is recycled to initiate another round of transcription. This is achieved through the action of multiple CTD kinases; including CDK7, of the TFIIH complex, and CDK9, of the Positive transcription elongation factor b (P-TEFb) complex; and their counterparts the CTD phosphatases, including SSU72 and CTDP1 (Jeronimo et al., 2013;Mayfield et al., 2016). Distinct pools of unphosphorylated and hyperphosphorylated RPB1 are detected in cell lysates using polyacrylamide gel electrophoresis (PAGE) due to a dramatic shift in the isoelectric point of RPB1 resulting from hyperphosphorylation. This physical characteristic of hyperphosphorylated RPB1 allowed for the initial discovery and characterization of the CTD and continues to be a useful tool in the study of CTD phosphorylation (Corden et al., 1985;Mayfield et al., 2019).
There are many ways to interrogate both the identity and abundance of modifications in CTD substrates, including antibodies (Jeronimo et al., 2013), mass spectrometry  and biophysical approaches like small-angle x-ray scattering (Portz et al., 2017). However, EMSA has the advantage of direct visualization, easy setup, and a rapid completion time without the requirement for specialized equipment. Here we treat GST-CTD fusion proteins with various CTD kinases, resolve reaction products in electrophoresis, and visualize product bands to describe reaction outcomes qualitatively. This approach is useful for verifying kinase and phosphatase activity against CTD substrates, processive/stochastic addition or removal of phosphates, and qualitative estimation of the number of phosphates added to CTD substrates. This approach is informative under both denatured/reduced and semi-native conditions allowing for tunable resolution and increasing downstream applicability in techniques like immunoblotting and band-excision coupled to mass spectrometry analysis.  3. Inoculate a single colony into 10ml of LB media containing 50 μg/ml kanamycin (10 ml LB + 10 μl 1,000x kanamycin stock) and grow overnight at 37 °C to generate a saturated culture.  18. Apply dialyzed protein to the prepared centrifugal filter unit and centrifuge the unit at 3,500 x g for 10-min intervals at 4 °C until the volume above the molecular weight cut-off filter reaches 1 ml. Transfer the volume above the filter to a 1.5 ml tube and centrifuge at 13,000 x g at 4 °C for 10 min to remove any protein aggregates. Transfer cleared supernatant to a fresh 1.5 ml tube. The stain can be reused multiple times.

Materials and Reagents
2. Rinse gel thoroughly in deionized water to remove residual Coomassie Brilliant Blue stain and cover gel completely with Destain. Incubate gel at room temperature with gentle agitation until stained protein bands become visible, and background is minimal, changing the destain as necessary.

Data analysis
The destained gel can be immediately visually interpreted. In SDS-PAGE applications, phosphorylation decreases the mobility of the GST-yeast CTD substrate. This is evidenced by a higher apparent molecular weight of the kinase treated sample relative to the no kinase control sample. In the semi-native PAGE, phosphorylation increases the mobility of the GST-yeast CTD substrate. This will appear as bands of lower apparent molecular weight in the kinase treated sample relative to the no kinase control. Gels can be imaged in any conventional gel imagine system,