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. 2001 May;21(9):3096-104.
doi: 10.1128/MCB.21.9.3096-3104.2001.

High-mobility-group proteins NHP6A and NHP6B participate in activation of the RNA polymerase III SNR6 gene

S Lopez  1 , M Livingstone-Zatchej, S Jourdain, F Thoma, A Sentenac, M C Marsolier
Affiliations

High-mobility-group proteins NHP6A and NHP6B participate in activation of the RNA polymerase III SNR6 gene

S Lopez et al. Mol Cell Biol. 2001 May.

Abstract

Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.

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Figures

FIG. 1
FIG. 1
Overexpression of the BRF1, NHP6A, or NHP6B genes rescues the thermosensitivity of snr6Δ2. MCM260 transformants containing either 2μm plasmids with the BRF1 (pFL44/BRF1), NHP6A (pFL44/NHP6A), or NHP6B (pFL44/NHP6B) genes, or the tetO-NHP6A or tetO-NHP6B constructs, or an empty vector (vector), were streaked on YPD plates and grown at 30 or 37°C for 3 days.
FIG. 2
FIG. 2
The transcription of mutant SNR6 genes is increased by overexpression of NHP6A or NHP6B. (A) The tetO-NHP6A and tetO-NHP6B constructs were introduced into the MCM260 strain, which contains only the snr6Δ2 allele. Transcripts derived from the snr6Δ2 genes were quantified in Northern blots by PhosphorImager analysis, using SNR31 transcripts as internal controls. (B) The Northern blot illustrates the transcriptional activation of SNR6 constructs harboring a 59-bp insert in the transcribed region and different mutations. These genes are borne by multicopy plasmids and generate transcripts (SNR6 maxi-RNA) easily distinguishable from the SNR6 RNA produced from the wild-type, chromosomal SNR6 gene. The steady-state levels of transcripts derived from the SNR6 maxigene constructs were analyzed in the wild-type strain YPH500α, with or without the overexpression of NHP6A or NHP6B, and quantified using SNR31 transcripts as internal controls. The transcription level of the wild-type (WT) maxigene construct without the overexpression of NHP6A or NHP6B (lane 1) was arbitrarily assigned the value 100%.
FIG. 3
FIG. 3
The transcriptional activity of wild-type and mutant SNR6 genes is reduced in the absence of NHP6A and NHP6B at 30°C. The transcription of the wild-type (WT), chromosomal SNR6 gene (A) and of SNR6 maxigene constructs either wild type or harboring different mutations (B) was monitored by Northern blotting in wild-type (WT, Y865 [8]) and nhp6AΔ nhp6BΔ mutant (Y869 [8]) cells. The steady-state levels of SNR6 RNA or maxi-RNA were quantified by PhosphorImager analysis, with SNR31 transcripts as internal controls. The transcription level of the wild-type maxigene construct in the wild-type strain was assigned the value 100%.
FIG. 4
FIG. 4
Protection over the TATA box of the SNR6 gene is dramatically altered in nhp6AΔ nhp6BΔ cells. Cultures of Y865 (NHP6A NHP6B) and Y869 (nhp6AΔ nhp6BΔ) were grown at 30°C (lanes 3 and 4 and lanes 7 and 8, respectively) and shifted to 37°C for 4 h (lanes 5 and 6 and lanes 9 and 10, respectively). Chromatin and genomic DNA were prepared and digested with different amounts of MNase. To display the cutting sites, the DNA was digested with PstI, fractionated on a 1% agarose gel, blotted to a nylon membrane, and hybridized to a probe close to the PstI site. Indicated are the nucleosome positions (white boxes), A and B blocks (black boxes), and TATA box (white oval) of the SNR6 gene. The marker (M) represents multiples of 256 bp and was hybridized separately. The protection of the TATA box (lanes 3 to 8) was lost when Y869 was shifted to 37°C (lanes 9 and 10).
FIG. 5
FIG. 5
NHP6B stimulates the transcription of wild-type and mutant SNR6 genes in vitro. NHP6B was added to in vitro transcription reaction mixtures containing cell extracts prepared from nhp6AΔ nhp6BΔ mutant cells (Y869 [8]) and different SNR6 templates, either wild type (WT) or mutated in the A or B block. The templates used were Bluescript derivatives harboring SNR6 wild-type or mutant genes. Reaction mixtures were incubated for 40 min at 25°C, and the transcription products were electrophoresed in a 6% polyacrylamide gel. The SNR6 transcripts were quantified by PhosphorImager analysis, and for each template, the basal level of SNR6 transcription in the absence of NHP6B was arbitrarily assigned the value of 1 unit.
FIG. 6
FIG. 6
NHP6B activates SNR6 transcription in a reconstituted system. NHP6B protein was added as indicated to in vitro transcription reaction mixtures containing either cell extracts (CE) prepared from nhp6AΔ nhp6BΔ mutant cells (Y869 [8]) or purified Pol III and recombinant TFIIIB (B) or Pol III, TFIIIB, and TFIIIC (B+C). A Bluescript-derived plasmid harboring the wild-type SNR6 gene was used as a template. Reaction mixtures were incubated for 40 min at 25°C, and the transcription products were electrophoresed in a 6% polyacrylamide gel. The SNR6 transcripts were quantified by PhosphorImager analysis, and the basal level of SNR6 transcription in the absence of NHP6B was arbitrarily assigned the value of 1 unit in each case.
FIG. 7
FIG. 7
NHP6A and NHP6B affect the transcript levels of several Pol III genes in vivo. (A) The steady-state levels of 5S RNA, tRNAHis, tRNAIle(UAU), and the transcripts derived from the SNR31 and SNR6 genes were analyzed by Northern blotting in the wild-type strain YPH500α (WT), with or without the overexpression of NHP6A or NHP6B (lanes 1 to 3), and in wild-type (lane 4, Y865 [8]) and nhp6AΔ nhp6BΔ mutant (lane 5, Y869 [8]) cells. The steady-state levels of the RNA were quantified by PhosphorImager analysis, and the quantity of each transcript was normalized with SNR31 transcripts as internal control. The relative RNA levels in the control wild-type strains (lanes 1 and 4) were arbitrarily assigned the value of 1 unit. (B) NHP6B was added to in vitro transcription reaction mixtures containing cell extracts prepared from nhp6AΔ nhp6BΔ mutant cells (Y869 [8]) and different templates containing either 5S rDNA, tDNAHis, tDNAIle(TAT), or the wild-type SNR6 gene as a control. The plasmids used are described in Materials and Methods. Reaction mixtures were incubated for 40 min at 25°C, and the transcription products were electrophoresed in a 6% polyacrylamide gel.
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