Abstract
Concomitant with RNA polymerase II (Pol II) transcription, RNA maturation factors are recruited to the carboxyl-terminal domain (CTD) of Pol II, whose phosphorylation state changes during a transcription cycle. CTD phosphorylation triggers recruitment of functionally different factors involved in RNA processing and transcription termination; most of these factors harbor a conserved CTD interacting domain (CID). Orchestration of factor recruitment is believed to be conducted by CID recognition of distinct phosphorylated forms of the CTD. We show that the human RNA processing factor SCAF8 interacts weakly with the unphosphorylated CTD of Pol II. Upon phosphorylation, affinity for the CTD is increased; however, SCAF8 is promiscuous to the phosphorylation pattern on the CTD. Employing a combined structural and biophysical approach, we were able to distinguish motifs within CIDs that are involved in a generic CTD sequence recognition from items that confer phospho-specificity.
Highlights
Serine residues can be phosphorylated during transcription elongation [11,12,13], and a varying phosphorylation pattern is generally believed to be the result of a balanced action of sitespecific carboxyl-terminal domain (CTD) kinases and phosphatases
By means of co-crystal structures, we further addressed the question of whether a CTD phosphorylated at position Ser-2 and Ser-5 can bind to SCAF8 in a -turn conformation and how and to which extent different phosphorylated and unphosphorylated forms of the CTD are recognized by the CTD-interacting domain (CID)
SCAF8-CID Binding to the CTD—Because different CIDs bind to different phospho-forms of the CTD, we asked whether there is a generic affinity of SCAF8-CID for the unphosphorylated form of the CTD
Summary
Cloning and Protein Preparation—DNA encoding amino acids 1–134 of the human SCAF8-CID was amplified by PCR and ligated into pET28b (Novagen) so that the expression construct obtained encodes an additional carboxyl-terminal hexahistidine tag. Selenomethionine incorporation into SCAF8-CID(N15M/Y17M) was performed as described in Van Duyne et al [40] using the protocol for cell growth and overexpression as for wild-type protein. The purification procedure of the selenomethionine-labeled SCAF8-CID(N15M/ Y17M) variant was performed as described for wild-type protein, except that a concentration of 5 mM dithioerythritol was used instead of 1 mM dithioerythritol. For cryo-protection, before flash-cooling in liquid nitrogen native, the SCAF8CID Ser(P)-2-CTD and selenomethionine-substituted protein crystals were transferred into their reservoir solution containing additional 20% (v/v) glycerol, whereas co-crystals of SCAF8-CID with Ser(P)-2/Ser(P)-7-CTD peptides were transferred into the reservoir solution containing increased ethylene glycol concentration (final 10% (v/v)). Phase extension to the wild-type, high resolution SCAF8-CID data set was performed following a rigid-body protocol [47] using the SCAF8CID(N15M/Y17M) model. Structure factor amplitudes and atomic coordinates are deposited at the Protein Data Bank under the entries 3D9I, 3D9J, 3D9K, 3D9L, 3D9M, 3D9N, 3D9O and 3D9P
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
