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1.
Figure 6

Figure 6. Termination factors Pcf11 and Nrd1 at 3’ ends. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A. Average distributions of Nrd1, S2-PO4, Rat1 and Pcf11 across long ORFs as in Fig. 1. Note coincident peaks of Nrd1, Pcf11 and Rat1 at 3’ ends (*) (see also Supp. Figs. 6E, F).
B–D. Plots of ChIP signals on selected genes showing discrete peaks of Nrd1 and Pcf11 accumulation (*) at poly (A) sites.
E. Average distributions of Nrd1, S2-PO4, Rat1, and Pcf11 across snoRNAs as in A.
F, G. Co-localization of Pcf11 with Nrd1 (*) on a snoRNA and a CUT.
H–J. Recruitment of Pcf11 and Nrd1 (*) to regulatory ncRNAs (red arrows) overlapping the 5’ ends of SER3, IMD2 and NRD1.
K, L. Effects of Pcf11 inactivation on transcription of SER3 and NRD1 with ncRNAs at their 5’ ends. Total pol II in WT (black) and pcf11-9 (PMY518) at 23° (red) and 37° (blue). Apparent readthrough transcription signals in pcf11-9 are marked with black arrows (see also Supp. Fig. 7E, F).

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.
2.
Figure 7

Figure 7. Pcf11 at centromeres, telomeres and pol III genes. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A–C. Average distributions of Pcf11 and pol II on all 16 centromeres (A, as in Fig. 1) and ChIP profiles of CEN1 and 4 with peaks of Pcf11 marked (*).
D. Average distributions of Pcf11, pol II, Rat1 and Nrd1 on tRNAs (275 genes, Supp. Table 2). Note higher pol II occupancy downstream of tRNAs than upstream (arrow).
E–G ChIP profiles with peaks of Pcf11 (*) over tRNAs and termination of convergent pol II transcription (E, F).
H. Pcf11 localization (*) on the pol III transcribed SCR1 gene for signal recognition particle RNA.
I, J Average distributions of Pcf11, pol II, Rat1, and Nrd1 at the telomeres with and without Y’ elements. Telomere sequences (dotted lines) and 1 kb of subtelomeric sequence (smooth lines) are shown.
K, L. ChIP profiles of Pcf11 and pol II on TEL7L (−Y’) and TEL8L (+Y’) (red bars). Chromosome ends are at left. Peaks of Pcf11 recruitment are marked (*).

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.
3.
Figure 1

Figure 1. Gene-specific CTD-PO4 dynamics in yeast. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A–C, G–I. Average distributions of total pol II (tot1, black), and phospho-CTD isoforms on highly transcribed short (<800 bases, 248 genes) medium (800–2000 bases, 462 genes) and long (>2000 bases, 128 genes) coding genes, snoRNAs, CUTs and SUTs (red arrows and dotted lines, Supp. Table 2). Transcription units for coding genes are as defined by RNA-seq50. X-axes show transcription units divided into 10 equal intervals (dotted line) with 1 kb of 5’ and 3’ flanking sequence (smooth line). Y-axes represent log2 ChIP signals relative to input DNA.
D–F, J–L. Average distributions of CTD phospho-isoforms normalized to total pol II. Note that the scale for the snoRNA plots differs from that for CUTs and SUTs.

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.
4.
Figure 4

Figure 4. A–G. Kin28 kinase enhances elongation at the 5’ ends of long genes. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A–C. Average distributions of total pol II across well-expressed short, medium and long genes (as in Fig. 1) in WT cells (PMY515, black) and the kin28as mutant (SHY508, red) treated with the inhibitor NP-PP1.
D–G. Plots of ChIP signals on selected genes reveal peaks of pol II accumulation (*) at 5’ ends of long (E–G) but not short genes (D). See also Supp. Fig. 2A–E.
H–L. Ctk1 kinase enhances elongation at the 3’ ends of a subset of genes.
H. Western blot of CTD S2-PO4 at time points after shifting the ctk1 tet-degron mutant (DBY739) to 37° + doxycycline (10 µg/ml) to deplete Ctk1. Pgk1 is a loading control.
I. Average distributions of pol II across 100 highly transcribed genes (Supp. Table 2) with 3' accumulation when Ctk1 is depleted in the ctk1tet-degron (red) relative top WT (black, DBY311). See also Supp. Fig. 2I.
J–L. Peaks of pol II accumulation (*) at 3’ ends after Ctk1 depletion. See also Supp. Fig. 2F.

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.
5.
Figure 2

Figure 2. A 450 base interval between S5 and S2 phosphorylation at 5’ ends regardless of gene length. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A–C. Plots of log2 Chip signals relative to input DNA and smoothed curves for total pol II (black) CTD S2-PO4 (green), S5-PO4 (red) and S7-PO4 (blue). Individual points are average ChIP signals at 20 base intervals. Red bars mark transcript boundaries 50. Note the delay between S5- and S2 phosphorylation (red and green arrows) is relatively constant on short (A) and long (B, C) genes. X-axes are nucleotide positions on the respective chromosomes.
D. Median ChIP signals (solid lines) and 50% central range (dashed lines) vs distance from the transcription start site (TSS) for 1349 coding genes (see Supp. Table 2). Arrow marks the cross-over of S2- and S5-PO4 plots
E. Distance from the TSS to the S2–S5 cross-over vs gene length for 438 coding genes (Supp. Table 2). Red dots are ribosomal protein genes. Note the distance to the cross-over is independent of gene length.
F–H. Plots of Chip signals on selected genes showing variable patterns of S7-PO4 distributions across genes.

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.
6.
Figure 5

Figure 5. Nrd1 co-localizes with S7-PO4 on ncRNA and mRNA genes. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A–C. Average distributions of Nrd1, S5-PO4 and S7-PO4 across CUTs (230 genes), SUTs (128 genes) and long ORFs (128 genes, Supp. Table 2 as in Fig, 1).
D. Nrd1 localization correlates better with S7-PO4 than with S5-PO4. Absolute values of Pearson’s correlation coefficients for ChIP signals of Nrd1 and S5-PO4 and Nrd1 and S7-PO4 were calculated throughout each gene and 1kb of 5’ and 3’ flanking sequence and plotted relative to one another for 246 highly expressed ORFs 1000–2000 bases long.
E–H. Peaks of Nrd1 and S7-PO4 (*) on an ORF (E), CUT (F), SUT (G) and an intronic snoRNA (H).
I. Apparent premature termination marked by loss of total pol II (arrow) and accumulation of Rat1 (*) downstream of the intronic snR54 in IMD4 (see also Supp. Fig. 5A–C).
J. An exceptionally short transcription unit (blue arrow, left panel) convergent with CLB5 (left panel) that corresponds to a stable unannotated transcript (red arrow, right panel). Right panel is a screenshot from http://steinmetzlab.embl.de/NFRsharing/ with tracks for nucleosome occupancy (green) and hybridization signals (blue heat map) for 5 microarray experiments. (See also Supp. Figs. 5D–G, 6I).
K. Nrd1 enrichment without pol II on ARSs. Average distributions of Nrd1 (black) and total pol II (green) across 209 functional ARS elements 51 (Supp. Table 2).

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.
7.
Figure 3

Figure 3. Enrichment of S7-PO4 on introns and variation of CTD-PO4 with transcription level and promoter structure. From: Gene-specific RNA pol II phosphorylation and the "CTD code".

A, B. Plots of Chip signals on selected genes showing enrichment of S7-PO4 on introns.
C. Average values of CTD S2, S5 and S7 phosphorylation normalized to total pol II on 282 introns (dotted lines) and flanking sequences (smooth lines, 1kb 5' and 3'). Note enrichment of S7-PO4 starting near 5’ splice sites (arrow).
D. Log2 ChIP signals within 123 genuine introns (red) from highly expressed genes and 919 decoy introns were calculated relative to 500 bases of upstream and downstream flanking sequence (Wilcoxon rank sum test P value < 2.2 × 10−16). The decoy introns were extracted from highly expressed intronless genes based on length and positions that match the population of genuine introns.
E, F. Average values of CTD phosphorylation on S2, S5 and S7 normalized to total pol II on ORF’s of similar length (800–1200 bases) in Group 1 (174 genes) and Group 3 (324 genes) with low and high transcription levels respectively 25 (Supp. Table 2) as in Fig. 1. Note that on average pol II on highly expressed genes has higher S5-PO4 and lower S2-PO4 and S7-PO4 than on poorly expressed genes.
G, H. Average values of CTD phosphorylation on S2, S5 and S7 normalized to total pol II on genes with occupied (OPN, 544 genes) vs depleted (DPN, 493 genes) proximal nucleosome promoter structures 27. Note higher S7-PO4 and lower S5-PO4 on DPN genes.

Hyunmin Kim, et al. Nat Struct Mol Biol. ;17(10):1279-1286.

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