A Tandem SH2 Domain in Transcription Elongation Factor Spt6 Binds the Phosphorylated RNA Polymerase II C-terminal Repeat Domain (CTD)

Abstract

Spt6 is an essential transcription elongation factor and histone chaperone that binds the C-terminal repeat domain (CTD) of RNA polymerase II. We show here that Spt6 contains a tandem SH2 domain with a novel structure and CTD-binding mode. The tandem SH2 domain binds to a serine 2-phosphorylated CTD peptide in vitro, whereas its N-terminal SH2 subdomain, which we previously characterized, does not. CTD binding requires a positively charged crevice in the C-terminal SH2 subdomain, which lacks the canonical phospho-binding pocket of SH2 domains and had previously escaped detection. The tandem SH2 domain is apparently required for transcription elongation in vivo as its deletion in cells is lethal in the presence of 6-azauracil.Spt6 is an essential nuclear protein (1, 2) that is required for transcription elongation together with Spt4 and Spt5 (3, 4). Spt6 co-localizes with Spt5 on transcribed genes in Drosophila polytene chromosomes (5, 6). Spt6 stimulates the elongation rate of RNA polymerase II in vivo (7). There is strong evidence that Spt6 is a histone chaperone. In cells lacking functional Spt6, transcription can start from cryptic promoters within the coding regions of genes (8), and transcription reinitiation from regular promoters is facilitated (9). This suggests that Spt6 is required for the correct positioning of nucleosomes in transcribed regions. Consistently, Spt6 interacts with histone H3 and can deposit nucleosomes on DNA (10). Spt6 is further involved in mRNA 3Ј-end formation (11) and export (12).The C-terminal region of mouse Spt6 binds the Ser-2-phosphorylated CTD, 2 suggesting a mechanism for how Spt6 is recruited to elongating RNA polymerase II (12). The Spt6 C-terminal region was predicted to contain an SH2 domain. We recently reported the crystal structure of this SH2 domain (13), which is the only SH2 domain in the yeast genome (14). The structure contained residues 1251-1349 of Candida glabrata Spt6 and showed a fold that combined features of both SH2 domain subfamilies present in higher eukaryotes (15, 16), likely resembling a common evolutionary ancestor of all SH2 domains (13). The structure also revealed a shallow phospho-binding pocket that explained the unusual binding specificity for phospho-serine rather than phospho-tyrosine. Our previous work, however, also showed that the SH2 domain alone did not bind CTD phospho-peptides (13), although a more extended C-terminal region does (12).To resolve this discrepancy, we investigated the structure and CTD binding of C-terminal fragments of Spt6 encompassing the SH2 domain and additional flanking regions. We found that a second SH2 domain is present directly C-terminal of the known SH2 domain. X-ray crystallography revealed that the two SH2 domains form an extended interface and are arranged in a previously unobserved manner into a rigid "tandem SH2 domain." In contrast to the previously studied single SH2 domain, the tandem SH2 domain binds Ser-2-phosphorylated CTD peptides in vitro. The tandem SH2 domain is apparently also required for t...

Cap Completion and C-Terminal Repeat Domain Kinase Recruitment Underlie the Initiation-Elongation Transition of RNA Polymerase II

“…4; see also Fig. S4B in the supplemental material), the last of which binds the S2-phosphorylated CTD in vitro (57). Moreover, genome-wide occupancy of Elf1 and Spn1 correlates with S2 phosphorylation levels (21).…”

“…This facilitates recruitment of the Paf complex (24,50,52,63), which establishes histone modifications linked to active transcription (64). Ctk1 phosphorylates S2 residues of the CTD further downstream (24,25) and stimulates Pol II association with elongation, chromatinmodifying, and RNA-processing factors (57,65,66), resulting in a productive elongation complex. Because recent evidence indicates that Bur1 is the P-TEFb (Cdk9) homologue (31), the same events likely occur during the initiation-elongation transition in higher cells.…”

Chromatin and Transcription in Yeast

“…Among its domains are tandem SH2 domains at its carboxy-terminal end, the only SH2 domains in S. cerevisiae (Maclennan and Shaw 1993;Dengl et al 2009;Diebold et al 2010b;Sun et al 2010;Close et al 2011;Liu et al 2011). The SH2 domains are required for Spt6 to interact with the CTD of Rpb1 of RNAPII, for normal levels of Spt6 recruitment to chromatin in vivo, and for wild-type function (Dengl et al 2009;Diebold et al 2010b;Mayer et al 2010;Sun et al 2010;Close et al 2011;Liu et al 2011).…”

The Composition of the Arabidopsis RNA Polymerase II Transcript Elongation Complex Reveals the Interplay between Elongation and mRNA Processing Factors

“…This tendency is visible in the magnified panels on the right ( Figure 3) and in the comparable merged images (Supplemental Figure 3), and it is also in line with the quantification of the colocalizing fluorescence signals (Supplemental Table 1). SPT6 from human and the fungus Candida glabrata binds preferentially to Ser2-phosphorylated RNAPII-CTD repeats (Sun et al, 2010;Yoh et al, 2007), and because of the colocalization of Arabidopsis SPT6L with the elongating RNAPII, we examined its interaction with the RNAPII-CTD. The interaction of the putative CTD-interaction domain SPT6L(Phe1218-Asp1412) with RNAPII-CTD peptides was examined by microscale thermophoresis (MST).…”