Structure of the Spt6 tandem SH2 domain. A, domain architecture of Spt6 (41) and alignment of the tandem SH2 domain amino acid sequence with corresponding regions in other eukaryotic Spt6 sequences (C. g., C. glabrata; S. c., S. cerevisiae; S. p., Schizosaccharomyces pombe; D. m., Drosophila melanogaster; H. s., Homo sapiens). Secondary structure elements are indicated above the sequences (cylinders, α-helices; arrows, β-strands). SH2-N is colored in blue, whereas SH2-C is in magenta. Invariant residues are in green, whereas conserved residues are in orange. Residues in the hydrophobic core are marked with triangles. HtH, helix-turn-helix domain, binds to double-stranded DNA; YqgFc, predicted to be a resolvase or ribonuclease, but in Spt6, catalytic residues are exchanged, thus probably not active; HhH, triple-helix-domain, binding to double-stranded DNA; S1, RNA-binding domain (13, 41). The sequence alignment of Spt6 tandem SH2 domains was obtained with ClustalW (42). B, ribbon representation of the Spt6 tandem SH2 domain structure. Subdomains are colored according to A. The left and right views are related by a 180° rotation around the vertical axis. C, superposition of SH2-N and SH2-C; structures were aligned by DALI-Lite (43), resulting in a root mean square deviation of 2.1 Å for 83 residues. The subdomains are colored according to A.

The tandem SH2 domain binds the RNA polymerase II CTD in vitro. A, fluorescence anisotropy titration of the CTD-derived phospho-peptide P1 (SYpSPTSPSYpSPTSPS; pS, phospho-serine) with different Spt6 tandem SH2 domain variants (open circles, wild-type tandem SH2 domain; open triangles, variant R1281K; filled circles, variant K1434A; filled triangles, variant R1281K/K1434A). The titration with the variant R1281K/K1434A could not be fitted with a simple binding model. The solid lines represent non-linear least square fits. B, Spt6 tandem SH2 domain surface conservation. Invariant and conserved residues are colored according to Fig. 1A. Indicated with circles are pocket 1 in SH2-N, the region that would correspond to pocket 1 in SH2-C, and the CTD-binding crevice in SH2-C. Residues Arg-1281 and Lys-1434 are represented as sticks and are labeled. The left and right views are related by a 120° rotation around the vertical axis. C, surface charge distribution. The domain surface is color-coded according to the electrostatic potential from negative (red) to positive (blue). Electrostatic surface potentials were calculated with APBS. The left and right views are related by a 120° rotation around the vertical axis.

Comparison with other SH2 domain tandem arrangements. The SH2 domains of ZAP-70 (Protein Data Bank (PDB) code 2oq1), SHP-1 (PDB code 2b3o), and SHP-2 (PDB code 2shp) are shown, with their N-terminal and C-terminal SH2 domains in blue and magenta, respectively. Additional structural elements between both SH2 domains are represented in orange, and a bound phospho-peptide in ZAP-70 is in red. The N-terminal SH2 domains in these proteins were superimposed onto Spt6 subdomain SH2-N.

The SH2 tandem domain is apparently involved in transcription elongation in vivo. Cultures of wild-type and Spt6 tandem domain deletion yeast strains (13, 31) were grown in YPD medium at 30 °C overnight and diluted to an optical density at 600 nm of 5 with fresh medium. The same amount of cells was spotted on plates in 10-fold serial dilutions. Plates were incubated for 5 days at 30 °C and inspected daily. 6-AU was added to SD-Ura plates at a concentration of 50 μg/ml.