Merge pull request #66 from ferhtaydn/fix/results-on-updated-dataset

results are updated for updated data set

files/annotator2_17/FN_annotations.txt

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+0114
+Crystallization trials were conducted for various forms of CgE, gE, and gE-gI (including CgE [residues 213–390], gE [residues 21–419], gE2 [residues 21–390], gE-gI [gE plus gI residues 21–266], gE2-gI2A [gE2 plus gI residues 21–208], and gE2-gI2B [gE2 plus gI residues 21–201]) both alone and complexed with wtFc or heterodimeric Fc. The only isolated protein to crystallize was CgE (described above), and the only complex of the six possible gE-gI/Fc complexes that crystallized was one that contained gE residues 21–419 and gI residues 21–266 and wtFc (residues 223–447). The complex crystals grew from drops containing a 2:1 molar ratio of gE-gI and wtFc mixed with an equal volume of well solution (0.1 M MES [pH 6.0] or 0.1 M HEPES [pH 7.0] and 0.9–1.1 M sodium malonate), resulting in a final pH of approximately 7.5. Microseeding increased the reproducibility of crystal growth.
+0114
+(C) Stereo superposition of the crystallographically determined CgE/Fc complex and the complex predicted with RosettaDock [34] using the structures of CgE and Fc.
+0077
+These interacting amino acids were also observed in the NMR titration experiments. In hTFIIEα AC-D, the NMR signals of E386, F387, E388, E389, V390, A391 and D392 were changed significantly upon addition of p62 PH-D (Supplementary Figure 1B) and also in p62 PH-D the NMR signals of K19, Q53, K54, I55, S56, E58, K60, A61, I63, Q64, L65, Q66, T74, T75 and F77 were changed by adding hTFIIEα AC-D (Supplementary Figure 2B).
+0077
+To investigate whether the STDE is involved in the binding, we prepared a longer construct (residues 351–439) containing both acidic regions and performed the NMR titration experiment under the same conditions (Supplementary Figure 4). The result was that the NMR signals of STDE showed no significant changes and the Kd of 400±43 nM was almost the same as that estimated using hTFIIEα AC-D.
+0077
+The binding site of hTFIIH p62 PH-D was localized to the second β-sheet (S5, S6 and S7), the loops between S1 and S2 and between S5 and S6 and the C-terminal H1 helix, where a substantial positive cluster is formed. Therefore, it is reasonable to speculate that the N-terminal highly acidic tail of hTFIIEα AC-D strongly binds to the positively charged surface of hTFIIH p62 PH-D. This is supported by the result that the binding is strengthened by removing NaCl from the buffer in the NMR titration experiments.
+0018
+DSL-1, OSM-11, LAG-2 extracellular domain (LAG-2Ex), EGL-17, or LIN-3 was fused to the GAL4 DNA binding domain (DB); the first six LIN-12 EGF repeats were fused to the GAL4 activation domain (AD). Pairwise interactions were tested with the yeast two-hybrid assay; positive interactions are indicated by blue staining. Both Notch DSL ligands and OSM-11 interacted with LIN-12 EGF repeats, whereas no interaction of LIN-3 EGF or EGL-17 FGF with LIN-12 Notch receptor EGF repeats was detected. LIN-12::DB fusion proteins exhibited strong self-activation (unpublished data); therefore, reciprocal fusions were not tested. Interaction controls are: (1) empty vectors; (2) DB-pRb and AD-E2F; (3) DB-Fos and AD-Jun; (4) Gal4p and pPC86; and (5) DB-DP1 and AD-E2F1.
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+To exclude the possibility that the abnormal cross-linking of basal transcription factors in ctk1Δ cells was due to issues with the specific antibodies, the experiment was repeated with strains carrying TAP-tagged Tfg1, Tfg2, or Tfg3 (all subunits of TFIIF). ChIP was carried out with IgG-agarose as described previously (Kim et al, 2004a). Again, spreading of TFIIF from the promoter throughout the transcribed region was seen in ctk1Δ cells (Figure 2, Tfg1–TAP result shown is representative). Some promoter preference was still seen, but the qualitative difference upon Ctk1 deletion was clear.
+0402
+Ctk1 is necessary for the dissociation of basal transcription factors from elongating RNA polymerase II (RNApII). (A) Schematic of the PMA1, ADH1, and PYK1 genes. The UAS of each gene is indicated by an open box (see Figure 3C). The TATA/promoter region and open reading frames are represented by black and grey boxes, respectively. Arrows indicate the position of the major polyadenylation sites reported previously (Kim et al, 2004a) and bars below the genes show the relative positions of PCR products in ChIP analysis. (B) Occupancy of Rpb1 and basal transcription factors (Sua7, TBP, Kin28, Tfb1, Tfa2) at the indicated regions in WT (YSB726) or ctk1Δ (YSB854) cells. INPUT was used to normalize the PCR amplification and the asterisk marks a non-transcribed PCR fragment indicated in all reactions as a background control. (C) Quantitation of the ChIP experiments in (B), with PMA1 as representative. The x axis indicates the specific primer pair used in each PCR. The y axis shows the specific signal relative to the negative control (i.e., a ratio of one is equivalent to background).