Nhp6, an HMG1 Protein, Functions in SNR6 Transcription by RNA Polymerase III in S. cerevisiae  Michael Kruppa, Robyn D Moir, David Kolodrubetz, Ian M Willis  Molecular Cell  Volume 7, Issue 2, Pages 309-318 (February 2001) DOI: 10.1016/S1097-2765(01)00179-4

Figure 1 Chromosomal Maps and Phenotypes of Clones that Suppress the Conditional Growth Defect of an nhp6ΔΔ Strain (A) Open reading frames in the region of chromosome VII that were found in the initial seven suppressor plasmids are marked by solid boxes and their gene designations. The thin lines show the regions present in a representative suppressor clone (pDK677) and two subclones. (B) Transformants of S. cerevisiae strain DKY625 that is deleted for NHP6A and NHP6B (designated nhp6ΔΔ) were grown at 30°C and 37°C. The indicated genes are present on high-copy number plasmids, except for PCF1–1, which is contained on a low-copy centromeric plasmid (see Experimental Procedures). The phenotype of one of the original suppressor clones, pDK677 (see [A]), is shown for comparison. (C) The solid boxes mark the genes found in the region of chromosome XII present on the suppressor clone pDK700. The regions present in three subclones are also indicated. (D) The indicated genes and subclones on high-copy number plasmids were transformed into strain DKY625 and grown at 30°C and 37°C as in [B] Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)

Figure 2 The Expression of Brf1 Is Unaffected in nhp6ΔΔ Cells at the Nonpermissive Temperature Mid-log phase cultures were grown as indicated to prepare cell lysates for Western blotting. (A) The untransformed nhp6ΔΔ strain (DKY625) and a transformant containing BRF1 on a high-copy number plasmid (DKY699, Figure 1A) were grown at 30°C. Cell lysates were probed with a polyclonal antibody to Brf1 (Sethy-Coraci et al., 1998). (B) The wild-type strain, DKY453, was grown in YEPD at 30°C and then shifted to 37°C. Cells were harvested before and after the temperature shift at the indicated times. Cell lysates from each time point were analyzed with the Brf1 antibody and, as a control for protein loading, with an antibody to TBP (Sethy-Coraci et al., 1998). (C) The nhp6ΔΔ strain (DKY625) was grown and analyzed as in (B) Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)

Figure 3 The Steady-State Level of U6 snRNA Decreases in nhp6ΔΔ Cells at Elevated Temperatures Yeast strains were grown to mid-log phase at 30°C and then shifted to 37°C. Cells were harvested before and after the temperature shift at the indicated times. RNA from each time point was used as a template for primer extension reactions with U4 snRNA– and U6 snRNA–specific primers. The extension products obtained using the primers individually are shown in the two rightmost lanes. U4 and U6 snRNA extension products are indicated by arrows. The asterisk marks a nonspecific extension product. RNA was analyzed from the wild-type (WT) strain (DKY453), the nhp6ΔΔ strain (DKY625), and the nhp6ΔΔ strain containing high-copy BRF1 or low-copy PCF1–1, as in Figure 1B Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)

Figure 4 Analysis of 5S rRNA and Bulk tRNA Synthesis in nhp6ΔΔ Cells at 37°C Wild-type and nhp6ΔΔ strains growing in low-phosphate YEPD medium were pulse-labeled for 5 min with [32P]orthophosphate at 25°C and at the indicated times after shifting to 37°C. (A) RNA (5 μg/lane) from the labeled cells was electrophoresed on a denaturing 10% polyacrylamide gel and visualized by staining with ethidium bromide. (B) The gel from (A) was dried and exposed to a PhosphorImager screen. (C) The synthesis of 5S rRNA and tRNA in the nhp6ΔΔ strain was determined relative to the wild-type strain at each time point. Integrated peak areas from (B) were normalized relative to the level of 5.8S rRNA in (A) Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)

Figure 5 Nhp6A Enhances In Vitro Transcription of SNR6 Multiple-round transcription reactions were performed using a yeast subcellular extract with 100 ng template DNA (2 μg/ml) at 15°C for 60 min. The extract, template, and NTPs were incubated for 10 min before addition of recombinant Nhp6A protein (100, 200, and 400 ng). Template DNAs include SNR6, tRNALeu3, sup3-e tRNASer-tRNAMet, and SUP4 tRNATyr genes. The U6 snRNA transcript is identified with an arrow. Transcripts in (A) were quantified, and their ratios relative to the samples containing no added Nhp6 protein are presented in (B). Control reactions (no Nhp6) were performed in duplicate (not shown in [A]), averaged, and plotted with the standard error Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)

Figure 6 Nhp6A Enhances TFIIIC-Dependent, but Not TFIIIC-Independent, Transcription on SNR6 (A) Single-round transcription on SNR6. Stalled ternary complexes containing a seven nucleotide nascent RNA transcript were formed on the SNR6 gene using a partially purified yeast extract (BRα fraction) in the absence (lanes 1 and 2) or in the presence (lanes 3–7) of Nhp6A protein. ATP was then added to allow elongation of the stalled RNA to full-length transcripts, and heparin (300 μg/ml) was also added to prevent reinitiation (lanes 2–7). Lanes 3–7 contain 50, 100, 250, 375, and 500 ng of Nhp6A, respectively. Reactions without Nhp6A were performed in duplicate (data not shown). (B) Quantitation of the data from (A) and from a multiple round experiment performed in parallel (not shown). Control reactions (no Nhp6) were performed in duplicate, averaged, and plotted with the standard error. (C) Multiple-round TFIIIC-dependent transcription of SNR6 and tRNALeu3 templates in a reconstituted system. Transcription complexes were assembled on template DNA, with TFIIIB components (recombinant Brf and TBP and yeast B′′) and TFIIIC for 10 min prior to the addition of Nhp6A (100, 250, and 500 ng). Following a 30 min incubation, NTPs and RNA polymerase III were added for an additional 30 min. (D) Multiple-round TFIIIC-independent (TATA box–dependent) transcription. Reactions were performed as in (C) but without TFIIIC and with 10-fold higher levels of TBP. (E) U6 snRNA transcripts from multiple (n ≥ 3) experiments were quantified and expressed relative to control reactions without Nhp6A Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)

Figure 7 Nhp6A Facilitates TFIIIC Binding to SNR6 (A) A native polyacrylamide gel of TFIIIC–DNA complexes formed on SNR6 (U6) and sup3-eST (tRNASer) templates in the presence and absence of Nhp6A. Template DNA (10 fmol) was incubated with purified yeast TFIIIC (2.5–10 fmol) for 10 min at 20°C. Nhp6A (0, 100, 250, 375, 500, and 750 ng) was then added (lanes 1–6 and lanes 7–12, respectively), and the incubation continued for 50 min. Nhp6A (750 ng) incubated with SNR6 and sup3-eST templates in the absence of TFIIIC is shown in lanes 13 and 14, respectively. (B) TFIIIC–DNA complexes from (A) were quantified and expressed relative to the average of duplicate reactions (data not shown) lacking Nhp6A Molecular Cell 2001 7, 309-318DOI: (10.1016/S1097-2765(01)00179-4)