Abstract
The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.
Highlights
TCERG1 interacts with hyperphosphorylated RNA polymerase II (RNAPII) C-terminal domain (CTD) through FF domains
Our study presents the first example of a nuclear factor requiring all three phosphoSer marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser7 phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events
It is important to note that the structures of individual FF domains of TCERG1 have been determined by solution NMR [34, 35], a recent crystallography study has shown that the three N-terminal FF domains (FF1–3) fold into a rigid integral structure with neighboring FF domains connected by a long helix [38]
Summary
TCERG1 interacts with hyperphosphorylated RNAPII CTD through FF domains. Results: We determined the structure of TCERG1 FF4 – 6 domain and its specific binding requirement of the CTD phosphoepitope.
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