In vivo binding and hierarchy of assembly of the yeast RNA polymerase I transcription factors

Abstract

Transcription by RNA polymerase I in Saccharomyces cerevisiae requires a series of transcription factors that have been genetically and biochemically identified. In particular, the core factor (CF) and the upstream activation factor (UAF) have been shown in vitro to bind the core element and the upstream promoter element, respectively. We have analyzed in vivo the DNAse I footprinting of the 35S promoter in wild-type and mutant strains lacking one specific transcription factor at the time. In this way we were able to unambiguously attribute the protections by the CF and the UAF to their respective putative binding sites. In addition, we have found that in vivo a binding hierarchy exists, the UAF being necessary for CF binding. Because the CF footprinting is lost in mutants lacking a functional RNA polymerase I, we also conclude that the final step of preinitiation-complex assembly affects binding of the CF, stabilizing its contact with DNA. Thus, in vivo, the CF is recruited to the core element by the UAF and stabilized on DNA by the presence of a functional RNA polymerase I.

Figure 1

Schematic representation of 35S RNA promoter in S. cerevisiae. Numbering is relative to the 35S transcriptional start (RIS). The ellipse indicates the first of five phased nucleosomes lying in the NTS. Positions of the most important DNA element are reported. The map position of the oligonucleotide (r3) used in this study for primer extensions is also reported (see text for details). ARS, autonomously replicating sequence.

Figure 2

In vivo footprinting of 35S RNA promoter. Different amounts (triangles) of DNAse I (3, 6, 12, 24 U) were introduced into purified nuclei of WT (NOY505) cells. The digestion profiles are compared with naked, deproteinized DNA (DNA) treated in vitro with different amounts of DNAse I (01, 0.2, 0.4 U). M, size marker (pBR322/MspI); lanes A and C, sequencing lanes. The schematic drawing indicates the area protected by the CF, the UAF, and Reb1p. Numbering starts from the RIS (+1).

Figure 3

Analysis of the CE region: comparison of NOY505 (WT), NOY558 (CF mutant), and NOY699 (UAF mutant). Starting from purified nuclei, chromatin was digested in vivo with 0, 6, 12, and 24 U of DNAse I, for 5 min at 0°C and in vitro and after deproteinization (DNA) with 0.1, 0.2, and 0.4 U of DNAse I for 2 min at 0°C; after DNA purification the samples were primer extended with the labeled oligonucleotide r3. (A) Comparison of WT (NOY505) and CF mutant (NOY558). DNA, in vitro treated samples; NOY505 and NOY558, in vivo treated samples. (B) Comparison of WT (NOY505) and UAF mutant (NOY699). DNA, in vitro treated samples; NOY505 and NOY699, in vivo treated samples. M, size marker (pBR322/MspI); lanes A and C, sequencing lanes. The schematic drawing indicates the CF putative binding site and relative map positions. Protected areas are shown by black arrowheads; enhanced cleavages are shown by white arrowheads; triangles indicate increasing amounts of DNAse I in the treatment; −, untreated DNA.

Figure 4

Analysis of the UPE region: comparison of NOY505 (WT), NOY558 (CF mutant), and NOY699(UAF mutant). Samples were treated as in Figure 3. (A) Comparison of WT (NOY505) and the CF mutant (NOY558). DNA, in vitro treated samples; NOY505 and NOY 558, in vivo treated samples. (B) Comparison of WT (NOY505) and the UAF mutant (NOY699). DNA, in vitro treated samples; NOY505 and NOY 699, in vivo treated samples. M, size marker (pBR322/MspI); lanes A and C: sequencing lanes. The schematic drawing indicates the UAF putative binding site and relative map positions. Protected areas are shown by black arrowheads; enhanced cleavages are shown by white arrowheads; triangles indicate increasing amounts of DNAse I in the treatment; −, untreated DNA.

Figure 5

Analysis of the CE region: comparison of NOY505 (WT), D128-1d (RPA43 mutant), and NOY604 (RRN3 mutant). Samples were treated as in Figure 3. (A) Comparison between WT (NOY505) and RPA43 mutant (D128-1d). DNA, in vitro treated samples (0.1 and 0.2 U of DNAse I); NOY505 and D128-1d, in vivo treated samples. (B) Comparison of WT (NOY505) and RRN3 mutant (NOY604). DNA, in vitro treated samples (0.1 and 0.2 U of DNAse I); NOY505 and NOY 604, in vivo treated samples. M, size marker (pBR322/MspI); lanes A and C: sequencing lanes. The schematic drawing indicates the CF putative binding site and relative map positions. Protected areas are shown by black arrowheads; enhanced cleavages are shown by white arrowheads; triangles indicate increasing amounts of DNAse I in the treatment; −, untreated DNA.

Figure 6

Analysis of the UPE region: comparison of NOY505 (WT), D128-1d (RPA43 mutant), and NOY604 (RRN3 mutant). Samples were treated as in Figure 3. (A) Comparison between WT (NOY505) and RPA43 mutant (D128-1d). DNA, in vitro treated samples (0.1 and 0.2 U of DNAse I); NOY505 and D128–1d, in vivo treated samples. (B) Comparison of WT (NOY505) and RRN3 mutant (NOY604). DNA, in vitro treated samples (0.1 and 0.2 U of DNAse I); NOY505 and NOY604, in vivo treated samples. M, size marker (pBR322/MspI); lanes A and C: sequencing lanes. The schematic drawing indicates the UAF putative binding site and relative map positions. Protected areas are shown by black arrowheads; enhanced cleavages are shown by white arrowheads; triangles indicate increasing amounts of DNAse I in the treatment; −, untreated DNA.

Figure 7

Analysis of the putative Reb1p binding site. DNA, samples were treated in vitro with increasing amounts of DNAse I (as in Figure 3). NOY505, D128-1d, NOY699, NOY558, and NOY604 samples were treated in vivo (as in Figure 3) with increasing amounts of DNAse I (triangles) in different mutant strains. The schematic drawing indicates the putative Reb1p-binding region.