Figure 1. Histone residues in the DNA entry-exit site of the nucleosome are important for transcription termination.

(A) Yeast strains contain either an SNR47 transcription termination reporter (top, KY3220) or a control transcription cassette lacking the SNR47 terminator (bottom, KY3219) integrated at the LEU2 locus. Black arrows denote the transcripts produced from each reporter in wild type (WT) and termination mutant backgrounds. (B) Yeast dilution assays to monitor growth of strains expressing the indicated H3 and H4 derivatives as the only source of H3 or H4. Library plasmids (TRP1-marked, CEN/ARS) (Nakanishi et al., 2008) expressing the histone gene mutations were introduced by plasmid shuffling into strains expressing the SNR47 termination reporter (KY3220; left) or the reporter control (KY3219; right). For each strain, a 10-fold dilution series (starting at 1 × 10⁸ cells/mL) was plated to SC-Trp as a growth control and to SC-His-Trp + 0.5 mM 3-aminotriazole (3-AT), a competitive inhibitor of the HIS3 gene product, to identify mutants expressing the HIS3 gene. Plates were incubated at 30°C for 5 days. (C) Diagrams of three snoRNA loci analyzed for termination readthrough by northern analysis. The black bar over each locus denotes the probe position. The intergenic SNR47 probe detects two read-through transcripts, as indicated by the long black and short blue arrows. The intergenic SNR48 probe detects a single readthrough transcript. For SNR13, the probe overlaps the downstream gene, TRS31, and detects a readthrough transcript of SNR13 (black), as well as the full-length TRS31 transcript (green). (D, F) Northern blot analysis to assess transcription readthrough of SNR genes in (D) H3 and H4 mutants (plasmids shuffled into KY812) and (F) H2A mutants (plasmids shuffled into KY943). Arrows correspond to those shown in the locus diagrams in panel C. SCR1 serves as the loading control. The northern blots are representative of three independent experiments. (E) X-ray crystal structure of the nucleosome denoting histone residues (highlighted in red) identified in the termination reporter screen and through northern analysis. Due to its buried location, H3 Q93 is not marked. H2A, H2B, H3, and H4 are colored in pink, yellow, lilac, and green, respectively. Structure from PDB ID 1ID3 (Luger et al., 1997).