Genome-wide location analysis of TFIIS and Pol II. (A) Enrichment of Pol II versus enrichment of TFIIS. The genome-wide enrichment of Pol II and TFIIS in YPD medium was assessed from ChIP–chip experiments using YGH2 (3HA-TFIIS) strain. A linear regression for Pol II enrichment versus TFIIS enrichment on class II probes and its equation are indicated. Red dots correspond to class III probes. (B) Quantitative ChIP analysis of TFIIS enrichment on selected genes. Immunoprecipitated fragments from ChIP experiments were amplified with primers as indicated in the Supplemental Material. The GAL1 ORF was used as a control. Error bars represent the standard deviation between at least three biological replicates. The background level was represented by a dotted line. (C) Quantitative ChIP analysis of TFIIS enrichment on rDNA. Immunoprecipitated fragments from ChIP experiments were amplified with primers as indicated in the Supplemental Material. The intergenic NTS1 and NTS2 regions were used as a control. Error bars represent the standard deviation between at least three biological replicates. The background level was represented by a dotted line. (D) Enrichment profile of Pol II and TFIIS on the PYK1 gene. The enrichment of Pol II (blue) and TFIIS (red) on PYK1 in YPD medium was assessed from ChIP–chip experiments using YGH2 (3HA-TFIIS) strain. The genomic positions of probe regions on chromosome 1 are indicated along the X-axis and represented by black points or circles. Watson strand-transcribed gene PYK1 is colored in blue. The enrichment ratio is indicated along the Y-axis.

Genome-wide location analysis of TFIIS and Pol III. (A) Enrichment of Pol III versus enrichment of TFIIS. The genome-wide enrichment of Pol III and TFIIS in YPD medium was assessed from ChIP–chip experiments with MW671 (3HA-RPC160) and YGH2 (3HA-TFIIS) strains, respectively. A linear regression and its equation are indicated. Red dots correspond to class III probes. (B) Enrichment profile of TFIIS on the SCR1 gene. The genome-wide enrichment of TFIIS in YPD medium was assessed from ChIP–chip experiments. The genomic positions of probe regions on chromosome 5 are reported along the X-axis and represented by black points. Watson strand-transcribed genes are colored in blue and Crick strand-transcribed genes are colored in green. The enrichment ratio is reported along the Y-axis.

Effect of rpb1-1 and rpc25-S100P mutations on TFIIS, Pol II, and Pol III occupancies on selected genes. Immunoprecipitations were performed using antibodies against 3HA (12CA5) for HA-TFIIS, 13Myc (9E10) for Rpc160-13Myc, and CTD (8WG16) for the Pol II Rpb1 subunit. Immunoprecipitated fragments from ChIP experiments were amplified with primers as indicated in the Supplemental Material. The GAL1 ORF was used as a control. The values are the average of three independent experiments. Error bars indicate the standard deviation. (A) TFIIS, Pol II, and Pol III occupancies in the rpb1-1 mutant. Standard ChIP assays were performed on chromatin prepared from the D788-4a strain transformed by RPB1-containing pYeB-B220 plasmid (wild type) or by empty vector Yep351 (rpb1-1). Cells were grown in selective SD medium complemented with amino acids at 30°C and then shifted for 30 min at 37°C. (B) TFIIS, Pol II, and Pol III occupancies in the rpc25-S100P mutant. Standard ChIP assays were performed on chromatin prepared from D792-3a strain transformed by RPC25-containing pRS315-RPC25 plasmid (wild type) or by empty vector pRS315 (rpc25-S100P). Cells were grown in selective SD medium complemented with amino acids at 30°C and then shifted for 10 h at 37°C.

Pol III, TFIIS, TFIIIB, and TFIIIC location analysis on the SCR1 gene. (A) Schematic organization of the SCR1 gene. The location of the PCR fragments amplified in ChIP analyses are indicated by gray and black lines. Black boxes represent TATA, A and B boxes, and Terminator (T). (B) Occupancy profile of Pol III, TFIIS, TFIIIB, and TFIIIC on the SCR1 gene. Standard ChIP assays were performed on chromatin prepared from YGH11 strain using antibodies against 3HA-TFIIS (12CA5) and Rpc160-13Myc (9E10), from MW4035 strain using antibodies against Bdp1-3HA (12CA5), from yOH1 strain using antibodies against Tfc1-13Myc (9E10), and from YGH15 strain using antibodies against Rpc11-3HA (12CA5). Cells were grown in YPD medium at 30°C. The GAL1 ORF was used as a control (c).

In vivo effect of TFIIS on Pol III transcription. (A) Growth of the YGH10 (SUP11 dst1-Δ ade2-1 ochre) strain relative to the MB159-2c (SUP11 DST1 ade2-1 ochre) strain. Cells were grown at 30°C for 3 d on minimal medium supplemented (+Ade) or not (−Ade) with adenine. (B) Growth of the dst1-null mutant at 30°C or 16°C. Suspensions of YPH500 (wild-type) or ESH1 (dst1-Δ) strains were serially diluted, spotted on YPD-rich medium, and grown for 3 d at 30°C or at for 5 d 16°C. (C) Growth of dst1 mutants at 30°C or 16°C. dst1-Δ (YGH12) strain was transformed by pRS425-pPGK vector, pRS425-DST1, pRS425-dst1R200A, or pRS425-dst1E291A plasmids bearing different DST1 alleles. Cells were grown in liquid SC-leucine medium at 30°C or shifted at time 0 at 16°C. (D) Northern blot analysis of tRNA₃^Leu abundance in wild-type and dst1-Δ strains. RNAs were extracted from wild-type (ESH1/pRS425-DST1) and dst1-Δ (ESH1/pRS425-pPGK) cells grown in SC-leucine medium at 30°C or shifted at 16°C for 8 h. tRNA precursors abundance was quantified, and fold decrease of transcription in dst1-Δ strain was compared with that in wild type grown at the same temperature (arbitrary set to 1). Average values of three independent experiments and standard deviations (SD) are indicated. (E) Analysis of the Pol III transcriptome at 16°C. RNAs were extracted from the dst1-Δ (ESH1) strain transformed by pRS425-pPGK vector, pRS425-DST1, pRS425-dst1R200A, or pRS425-dst1E291A plasmids grown at 30°C or shifted for 8 h to 16°C, and analyzed by hybridization to Pol III-specific microarrays. Expression log₂ ratios of class III genes in the mutant strains compared with wild type are presented according to the red–green color scale.

Pol II and Pol III genome-wide enrichment in TFIIS mutants at 16°C. The genome-wide enrichment of Pol III (A,C,E) or Pol II (B,D,F) in YPD medium (A,B) or SC-leucine medium (C–F) at 16°C was assessed from ChIP–chip experiments. A linear regression (red line) and its equation are indicated. The dotted line corresponds to y = x. Enrichment of Pol III or Pol II in dst1-Δ (YGH12) was compared with the wild-type strain (YGH11) (A,B). Enrichment of Pol III or Pol II in dst1-E291A (YGH12/pRS425-dst1E291A) (C,D) and dst1-R200A (YGH12/pRS425-dst1R200A) (E,F) strains was compared with the wild-type strain (YGH12/pRS425-DST1). (G,H) Quantitative ChIP analysis of TFIIIC and TFIIIB enrichment on selected class III genes. dst1-Δ and wild-type strains were grown in YPD medium at 30°C and shifted for 8 h to 16°C. Immunoprecipitations were performed using antibodies against 13Myc (9E10) for Tfc1-13Myc and 3HA (12CA5) for Bdp1-3HA. Immunoprecipitated fragments from ChIP experiments were amplified with primers as indicated in the Supplemental Material. The GAL1 ORF was used as a control. Error bars represent the standard deviation between at least three biological replicates.

TFIIS stimulates faithful Pol III transcription in vitro. (A) Effect of TFIIS on Pol III transcription. Standard in vitro Pol III transcription on the SUP4 template has been performed as described in the Materials and Methods, in the absence (lane 1) or presence of increasing quantities (0.1, 0.2, 0.4, 0.8, and 1.6 μg) of wild-type TFIIS (lanes 2-6) or mutant TFIIS–E291A (lanes 7–11) or with a mix of wild-type and E291A protein (M, lane 12). Transcription start sites are indicated. (B) Primer extension analysis of start sites used by Pol III on the SUP4 gene in vitro. Transcription reactions were performed in the absence of TFIIS, in the presence of wild-type TFIIS, mutant TFIIS–E291A, or with a B″ fraction. Transcription and primer extensions were performed as described in Materials and Methods using a probe hybridizing within the SUP4 intron. Positions of the major transcription start site are indicated. (C) Time-course analysis of RNA cleavage by Pol III. Pol III (lanes 1–14) or Pol IIIΔ (lanes 15–21) (Chedin et al. 1998) ternary complexes formed in the presence of 3XTPs were isolated on Sepharose CL-2B as described in the Supplemental Material and then incubated for 10 min with a 50 M excess of purified TFIIS (lanes 8–21) or without TFIIS (lanes 1–7). Ternary complexes were then incubated at 16°C in transcription buffer containing 5 mM MgCl₂ in the absence of nucleotides for various periods of time. The transcript sizes are indicated. Transcription with Pol IIIΔ results in the formation of an 18-mer RNA instead of a 17-mer for the wild-type enzyme (Chedin et al. 1998).