Interaction of TATA-binding protein with upstream activation factor is required for activated transcription of ribosomal DNA by RNA polymerase I in Saccharomyces cerevisiae in vivo

Abstract

Previous in vitro studies have shown that initiation of transcription of ribosomal DNA (rDNA) in the yeast Saccharomyces cerevisiae involves an interaction of upstream activation factor (UAF) with the upstream element of the promoter, forming a stable UAF-template complex; together with TATA-binding protein (TBP), UAF then recruits an essential factor, core factor (CF), to the promoter, forming a stable preinitiation complex. TBP interacts with both UAF and CF in vitro. In addition, a subunit of UAF, Rrn9p, interacts with TBP in vitro and in the two-hybrid system, suggesting the possible importance of this interaction for UAF function. Using the yeast two-hybrid system, we have identified three mutations in RRN9 that abolish the interaction of Rrn9p with TBP without affecting its interaction with Rrn10p, another subunit of UAF. Yeast cells containing any one of these individual mutations, L110S, L269P, or L274Q, did not show any growth defects. However, cells containing a combination of L110S with one of the other two mutations showed a temperature-sensitive phenotype, and this phenotype was suppressed by fusing the mutant genes to SPT15, which encodes TBP. In addition, another mutation (F186S), which disrupts both Rrn9p-TBP and Rrn9p-Rrn10p interactions in the two-hybrid system, abolished UAF function in vivo, and this mutational defect was suppressed by fusion of the mutant gene to SPT15 combined with overexpression of Rrn10p. These experiments demonstrate that the interaction of UAF with TBP, which is presumably achieved by the interaction of Rrn9p with TBP, is indeed important for high-level transcription of rDNA by RNA polymerase I in vivo.

FIG. 1

Locations of rrn9 deletions analyzed in TBP binding experiments and of rrn9 point mutations affecting the interaction with TBP in the two-hybrid system in vivo. Restriction enzyme sites used to construct various deletions are shown on the RRN9 gene at the top. Three regions containing rrn9 point mutations are indicated as I, II, and III, respectively, on the Rrn9p protein, and their amino acid sequences are shown, with amino acid residues altered by the mutations underlined. Rrn9p derivatives carrying the deletions indicated were labeled with [³⁵S]methionine, and their binding to GST-TBP was studied. Average values of binding, as percentages of input Rrn9p, obtained from three such experiments are shown for each construct. The results of one experiment are shown in Fig. 2.

FIG. 2

Binding of [³⁵S]methionine-labeled Rrn9p and its deletion derivatives to GST-TBP. Equivalent amounts of [³⁵S]methionine-labeled Rrn9p and its deletion derivatives were mixed with GST-TBP attached to glutathione-agarose beads for 30 min at room temperature. Beads were washed three times, and the labeled proteins bound to the beads were analyzed by SDS-PAGE; 10% of each of the input of the labeled proteins was also analyzed. An autoradiogram of the gel is shown. Positions of each intact labeled protein in the radioactive protein preparations used as input are shown by dots. Smaller radioactive proteins found in some of the input protein preparations, especially in Rrn9p and the Δ157-186 protein, may have been produced by incorrect translation initiation, premature termination of translation, or degradation of full-sized proteins during their preparation in reticulocyte lysate. In other similar experiments, binding of ³⁵S-labeled Rrn9p and its deletion derivatives to GST was analyzed in parallel to binding to GST-TBP. None of the labeled proteins were bound to GST control beads to any significant extent.

FIG. 3

Similarity of two regions of Rrn9p to ADs of some Pol II transcriptional activators. Regions I and II of Rrn9p (Fig. 1) are aligned to follow the conserved pattern published for ADs of p53 and VP16 (3, 7). Bulky hydrophobic amino acids used to align the sequences are boxed. Amino acid residues which have been shown to be important for activation by mutational analysis (, , , ; this study) are underlined. The position of the first amino acid of each sequence is shown. It should be noted that in the previous papers (3, 7), the amino acid sequences of the ADs of quite a few other transcription factors were also aligned with the sequences of VP16 (7) and p53 (3), and the boxed hydrophobic amino acids were suggested as a consensus sequence. Here, only the sequences of VP16 and p53, for which there is strong experimental support for the significance of these amino acids, are given.

FIG. 4

Suppression of the rrn9(F186S) mutant phenotype by fusion of the mutant gene to the SPT15 gene combined with overexpression of Rrn10p. Yeast strains were streaked on a synthetic glucose medium lacking tryptophan and leucine, and the plates were incubated at 30 and 36°C for 5 days. Positions of the strains on plates are indicated below the photograph. They are designated by numbers that follow NOY in the complete names. All strains carry the chromosomal rrn9Δ::HIS3 deletion and two plasmids in addition to pNOY103. One (CEN) is pRS314 (vector) or its derivatives carrying RRN9, rrn9(F186S), or the rrn9(F186S)-SPT15 fusion as indicated. The second plasmid (2 μm) is YEp351 (vector) or its derivative carrying RRN10 as indicated.

FIG. 5

Strategy used to screen for rrn9 mutations that abolish the interaction of Rrn9p with TBP in the yeast two-hybrid system. Structures of the DNA fragment generated after PCR mutagenesis (RRN9 PCR fragment) and the linearized form of pAS2 are shown as DBD-Rrn9p, indicating that a homologous recombination in vivo will form the structure identical to that of pNOY355, encoding Rrn9p fused to the DBD of Gal4p (DBD-Rrn9p). The structure of pNOY359 present in the reporter strain SFY526 is also shown (AD-TBP); pNOY359 carries a fusion gene encoding TBP fused to the AD of Gal4p. This strategy is based on that described in reference . For details of screening of mutants, see Materials and Methods.

FIG. 6

Suppression of rrn9(L110S/L269P) and rrn9(L110S/L274Q) mutant phenotypes by fusion of the mutant genes to the SPT15 gene, which encodes TBP. Strains were streaked on a synthetic glucose medium lacking tryptophan, and the plates were incubated at indicated temperatures for 3 days. All the strains carry the chromosomal rrn9Δ::HIS3 mutation and, in addition to pNOY103, a derivative of pRS314 carrying RRN9, rrn9 mutant genes, or the rrn9 mutant genes fused to SPT15, as indicated. The strains are indicated by numbers that follow NOY in the complete names.