Structure and activation mechanism of the yeast RNA Pol II CTD kinase CTDK-1 complex

Abstract

The C-terminal domain (CTD) kinase I (CTDK-1) complex is the primary RNA Polymerase II (Pol II) CTD Ser2 kinase in budding yeast. CTDK-1 consists of a cyclin-dependent kinase (CDK) Ctk1, a cyclin Ctk2, and a unique subunit Ctk3 required for CTDK-1 activity. Here, we present a crystal structure of CTDK-1 at 1.85-Å resolution. The structure reveals that, compared to the canonical two-component CDK-cyclin system, the third component Ctk3 of CTDK-1 plays a critical role in Ctk1 activation by stabilizing a key element of CDK regulation, the T-loop, in an active conformation. In addition, Ctk3 contributes to the assembly of CTDK-1 through extensive interactions with both Ctk1 and Ctk2. We also demonstrate that CTDK-1 physically and genetically interacts with the serine/arginine-like protein Gbp2. Together, the data in our work reveal a regulatory mechanism of CDK complexes.

Keywords: X-ray crystallography; cyclin-dependent kinase; mRNA processing; transcription.

Fig. 1.

Structure of a Ctk1•Ctk2•Ctk3 complex. (A) Schematic representation of the CTDK-1 subunits. (B) Identification of a catalytically active CTDK-1 core complex. GST-tagged yeast Pol II CTD (10 µM) was incubated with the indicated CTDK-1 complex (0.25 µM) and 5 mM ATP at 30 °C for 30 min. CTDK-1 activity was shown by a retarded band on SDS/PAGE, corresponding to phosphorylated GST-CTD. An asterisk denotes the stained band of Ctk1 or the Ctk1(D324N) mutant. (C) Crystal structure of the Ctk1∆N•Ctk2•Ctk3 complex determined at 1.85-Å resolution. Ctk1∆N, Ctk2, and Ctk3 are colored in cyan, yellow, and purple, respectively. The T-loop of Ctk1 is colored in orange. (D) Schematic diagram of the Ctk1∆N•Ctk2•Ctk3 complex.

Fig. 2.

Ctk3 stabilizes the T-loop of Ctk1 in an active conformation. (A) The conformation of the Ctk1 T-loop (orange) resembles that of an active CDK12•Cyclin K complex (PDB ID code 4NST). Key residues at the Ctk1–Ctk3 interface are shown as sticks. A citrate molecule (green) is located near T338. In CDK12•Cyclin K, selective residues, including the phosphorylated T893 (TPO893), are shown as white sticks. The nucleotide ADP•AlF₃ bound to CDK12 is colored gray. (B) Close-up view of the Ctk1 T-loop. (C) Schematics of the interactions between the Ctk1 T-loop and the Ctk3 H1 helix. (D) The interface between the CID-like domain of Ctk3 and Ctk1.

Fig. 3.

The Ctk2–Ctk3 interface. (A) The binding interface between the H2 helix of Ctk3 and Ctk2. (B) The binding interface involving the H3 helix of Ctk3. D278 of H3 forms a salt bridge with K330 on the T-loop of Ctk1. (C) Alignment of Ctk1∆N•Ctk2•Ctk3 and CDK2•Cyclin A (PDB ID code 1JST). Ctk1∆N•Ctk2•Ctk3 is colored as in Fig. 1C. CDK2 and Cyclin A are colored light cyan and white, respectively. The location of the H3 helix of Ctk3 resembles that of the N-terminal helix of Cyclin A (green).

Fig. 4.

A Ctk1–Ctk2 interface. Comparison of Ctk1∆N•Ctk2•Ctk3 (Left) and its human ortholog CDK12•Cyclin K (right, PDB ID code 4NST). Ctk2 inserts two strands (β′ and β′′) into the β-sheet of the Ctk1 N-lobe, forming an intermolecular β-sheet. Residues at the Ctk1–Ctk2 interface are shown as sticks.

Fig. 5.

Ctk1 interacts with the SR protein Gbp2 physically and genetically. (A) Ctk1•Ctk2•Ctk3 directly interacts with Gbp2. In vitro GST pull-down assays were performed with GST or GST-tagged Gbp2∆N and the Ctk1•Ctk2•Ctk3 complex as indicated. (B) Serial dilutions of yeast demonstrating a synthetic growth defect of ctk1∆ and gbp2∆ in yeast.