Structural basis for the recognition of the S2, S5‐phosphorylated RNA polymerase II CTD by the mRNA anti‐terminator protein hSCAF4

Abstract

The C‐terminal domain (CTD) of RNA polymerase II serves as a binding platform for numerous enzymes and transcription factors involved in nascent RNA processing and the transcription cycle. The S2, S5‐phosphorylated CTD is recognized by the transcription factor SCAF4, which functions as a transcription anti‐terminator by preventing early mRNA transcript cleavage and polyadenylation. Here, we measured the binding affinities of differently modified CTD peptides by hSCAF4 and solved the complex structure of the hSCAF4‐CTD‐interaction domain (CID) bound to a S2, S5‐quadra‐phosphorylated CTD peptide. Our results revealed that the S2, S5‐quadra‐phosphorylated CTD peptide adopts a trans conformation and is located in a positively charged binding groove of hSCAF4‐CID. Although hSCAF4‐CID has almost the same binding pattern to the CTD as other CID‐containing proteins, it preferentially binds to the S2, S5‐phosphorylated CTD. Our findings provide insight into the regulatory mechanism of hSCAF4 in transcription termination.

Blocker-SELEX: A Structure-guided Strategy for Developing Inhibitory Aptamers Disrupting Undruggable Transcription Factor Interactions

“…1b; Supplementary Fig. 1a) 53 . Consequently, the absence of specifically designed chemical tools has hindered in-depth exploration of the precise biological function associated with this interaction.…”

“…To determine the binding specificity of SCAF4_LS sequence to the PPI interface, critical residues on the SCAF4 interface were substituted, and their binding to SCAF4_LS was characterized using a fluorescence polarization assay. Three arginine residues (R23, R71, and R112), which are known to be involved in SCAF4 recognition of RNAP2 53 , were substituted with alanines, resulting in weaker interaction with SCAF4_LS and reducing the binding affinity by over two-fold (Fig. 2e; Supplementary Fig.…”

“…3n). SCAF4_LS sequence was recaptured in developing aptamers for SCAF8 SCAF8, a crucial paralog of SCAF4, shares a comparable CID domain with an overall RSMD of 0.266 Å and a PPI interface for RNAP2 recognition 53,54 (Supplementary Fig. 6a).…”

The Nrd1–Nab3–Sen1 transcription termination complex from a structural perspective

“…CTD-Pro3 interacts with a conserved Tyr in helix 4 and two hydrophobic side-chains in helix 7 ( Figure 2A,B ) and, in many complexes, the CTD Pro3 forms a β-turn. In general, peptide bonds of CTD-Pro3/6 adopt the trans configuration (SCAF4 and SCAF8 complexes included [ 62 , 63 ]) , with the exception of the Nrd1 CID/CTD-pSer5 complex where the pSer5-Pro6 bond of the first repeat is in cis , causing a strong kink [ 59 ]. A similar case was found in the Ssu72/CTD-pSer5 complex [ 64 , 65 ].…”