1. Volume 31, Issue 5, Pages 671-682 (September 2008)
Futile Cycle of Transcription Initiation and Termination Modulates the Response to Nucleotide Shortage in S. cerevisiae 
Marilyne Thiebaut, Jessie Colin, Helen Neil, Alain Jacquier, Bertrand Séraphin, François Lacroute, Domenico Libri 
Molecular Cell 
Volume 31, Issue 5, Pages (September 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
The URA2 Locus Contains a Cryptic Unstable Transcript that Overlaps with the 5′ End of the URA2 Transcript
(A) Scheme of the URA2 genomic locus. The URA2 mRNA (containing an intron) and the usURA2 CUT are represented by a gray line and a thick, black line, respectively. The heterogeneous 3′ ends of the CUT are represented by a dashed line. The transcription start sites for usURA2 (TSSCUT) and the URA2 mRNA (TSSmRNA) are indicated by a black and a white box, respectively. The region between the TSS (R box) is represented by a gray box. The position of the starting nucleotides are indicated (numbering is relative to the URA2 ATG). Arrows indicate the positions of the oligonucleotides used for RNaseH digestion and primer extension. The position of the probe used for northern blot analyses is indicated.
(B) Left panel: northern blot analysis (total and oligo dT-selected RNAs) of usURA2 transcripts in WT and degradation defective strains rrp6Δ and trf4Δ. The URA2 mRNA is not resolved in these electrophoretic conditions (5% PAGE). Right panel: the usURA2 CUT is sensitive to termination by the Nrd1p pathway. NRD1 is under control of the glucose-repressible GAL1 promoter in the Pgal-NRD1/rrp6Δ strain. Nrd1p was depleted by growth in glucose for 7 hr. The short unstable RNAs disappear to the profit of a longer species that extends in the URA2 gene and is not resolved in these conditions.
(C) Identification of the transcription start sites of the usURA2 and URA2 transcripts by RNaseH cleavage and northern blot analysis. RNaseH cleavage was performed with the 1046 or 1096 oligonucleotides as indicated. Bands derived from transcripts initiated at the upstream (TSSCUT) or downstream (TSSmRNA) start sites are indicated as usURA2 or URA2, respectively. Detection of RPS28A mRNA is used as loading control in (B) and (C).
(D) Primer extension of transcripts prepared from cells grown either in uracil-replete conditions or in the presence of 6-AU as indicated. Primer 1096 was used for extension. An extension product unrelated to URA2 served as loading control. The increase in URA2 mRNA and usURA2 CUT levels upon uracil depletion is indicated below the gel. Amounts are normalized to loading and expressed relative to the WT and trf4Δ strains under uracil-replete conditions for the mRNA and the CUT, respectively.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Expression of usURA2/URA2 Locus Depends on a Single Promoter
(A) Northern blot analysis of RNaseH-cleaved transcripts derived from constructs bearing mutations in two putative TATA boxes (TATA1 and TATA2, positions indicated in the scheme). The different constructions are ectopically expressed in strains that are deleted for the 5′ region of the endogenous URA2 (including the CUT). In these conditions, omission of uracil from the growth medium is sufficient to activate downstream initiation (i.e., the addition of 6-AU is not required). μ-tata1 and μ-tata2 indicate mutation of TATA1 and TATA2, respectively. Detection of U4snRNA is used as a loading control.
(B) Expression of the usURA2/URA2 locus under control of the GAL1 and Tet-off heterologous promoters. Left panel: the GAL1 promoter (thick arrow, black) was inserted upstream of the genomic usURA2/URA2 locus. The usURA2/URA2 TATA box is driving expression of the gene (inserted sequences do not include the GAL1 TATA box). Cells were cultivated in the presence of glucose or galactose to repress or activate, respectively, the GAL1 promoter and under saturating uracil or in the presence of 6-AU to regulate TSS selection. RNaseH northern blot analysis is as in Figure 1. Detection of U6snRNA was used as a loading control. Right panel: usURA2/URA2 5′ fragment spanning from the first CUT start site to the −1 nucleotide relative to the ATG was cloned upstream of the CUP1 gene (not shown in the scheme) under control of a tetracycline-repressible promoter (Tet-off, thick gray arrow) in plasmid pCM185. The Tet-off promoter is based on a CYC1 core promoter containing a TATA box and CYC1 start sites. The latter have been precisely replaced with the TSSCUT. Note that, in this construct, expression is driven from the CYC1 TATA box. A thick gray line indicates CYC1 sequences in the scheme. Ectopic expression of this construct was as in (A) with the addition of doxycycline to repress the Tet-off promoter as indicated. Primer extension analysis was performed as in Figure 1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The Presence of the CUT Downregulates Expression of the mRNA
(A) Scheme of the different mutants analyzed.
(B) Northern blot analysis of RNaseH cleaved transcripts (oligonucleotide 1096) from the UpΔ2 strain under uracil-replete (left panel) and uracil-shortage (right panel) conditions as in Figure 1D. The two panels are from the same gel. Note that all transcripts are initiated at TSSCUT and at additional downstream sites likely due to the deletion of the major TSSmRNA. Detection of RPS28A mRNA is used as a loading control. The mRNA levels are calculated relative the WT construct in both growth conditions.
(C) Northern blot analysis of RNaseH cleaved transcripts derived from ectopically expressed μ-TSSCUT construct, containing a mutation of all CUT start sites. Growth conditions are as in Figure 2A. The CUT and the mRNA are indicated as in Figure 1. Detection of U6snRNA mRNA is used as a loading control.
(D) Northern blot analysis of RNaseH cleaved transcripts derived from the usURA2/URA2 locus in rpb1-S1401P and rpb1-S1401P/trf4Δ cells. Detection of RPS28A mRNA is used as a loading control.
(E) The rpb1-S1401P mutation affects transcription start site selection of the ADH1 gene. Primer extension of ADH1 transcripts from WT and rpb1-S1401P cells. The numbering corresponds to the position of the start site relative to the ATG of ADH1.
(F) URA2 mRNA expression is activated in transcription initiation-defective mutants. Northern blot analysis of RNaseH cleaved transcripts (oligonucleotide 1096) from the strains indicated in a trf4Δ background to visualize both the CUT and the mRNA (duplicate lanes). The sua8-4 mutant corresponds to the rpb1-G1388 allele of the large RNA Pol II subunit. sua7-1 is an allele of the general transcription factor TFIIB. For comparison, the rpb1-S1401P strain and its isogenic WT (W303) are analyzed in parallel. All cells are grown at the permissive temperature (30°C) for the mutants. Detection of U6snRNA is used as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Mutational Analysis of the CUT and mRNA TSS
(A) Schematic drawings of the constructs analyzed.
(B) Northern blot analysis of RNaseH cleaved transcripts derived from constructs bearing duplication of the CUT or mRNA start sites in WT and trf4Δ cells. Ectopically expressed constructs under the conditions are indicated as in Figure 2A. Analysis of a WT construct in trf4Δ cells under both growth conditions is shown for comparison in lanes 1 and 2 (same gel). Detection of U6snRNA is used as a loading control. The asterisks indicate additional start sites downstream of the duplicated TSS.
(C) Northern blot analysis of RNaseH cleaved transcripts derived from constructs in which the TSSCUT have been mutated or replaced with weak heterologous start sites derived from the ADH1 and the SNR14 genes. Analysis is as in (B).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Mutational Analysis of the R Box
(A) Relevant sequences of the constructs analyzed. Mutation indicated by lowercase characters. The Nab3p and Nrd1p binding sites are underlined. In Rbox-Rev and Rbox-Inv, the R box was replaced, respectively, with its reverse complement and with its inversed derivative (same sequence read 3′–5′). In Rbox-NoNab3, all the Nab3p sites have been mutated without altering the T-content of the sequence. In Rbox-Nrd1 all the Nab3 sites have been mutated to Nrd1p sites, and the T-rich symmetrical motif was also changed.
(B) Northern blot analysis of RNaseH cleaved transcripts-derived R box mutants. All constructs were expressed ectopically and analyzed as in Figure 2A. Loading control as indicated.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 The URA8 Locus Is Regulated Similarly to the usURA2/URA2
(A) Scheme of the URA8 gene. The transcription start sites of the CUT (usURA8) and the mRNA (URA8) are indicated by a black box and a white box, respectively. The starting nucleotides as determined by primer extension are also indicated. The position of the oligonucleotide used for primer extension (1204) is shown by an arrow. The usURA8 and URA8 transcripts are indicated by black lines and thick, gray lines, respectively. Heterogeneity in the 3′ end of usURA8 is represented by a dashed line.
(B) Primer extension analysis of usURA8 and URA8 transcripts under the conditions and in the strains indicated. CSM, complete synthetic medium, repressing conditions. Growth in the presence of 6-AU activates production of the URA8 mRNA (compare lanes 1 to 2 and 3 to 4) presumably because CTP depletion occurs as a consequence of UTP depletion.
(C) Primer extension analysis of transcripts derived from the ADE12 locus in the indicated strains. The genomic organization is similar to the URA2 and URA8 loci. usADE12 and ADE12 indicate, respectively, the CUT and the mRNA. The reason why the addition of 6-AU activates use of the TSSmRNA (compare lanes 1 and 2 to 3 and 4) is unclear.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Models to Explain the Regulated Selection of the TSSmRNA under Uracil Shortage
Both models are consistent with the observation that the CUT levels do not change when uracil is limiting in spite of a considerable increase in mRNA production and that both species depend on the activity of a single promoter.
(A) The R box promotes internal Pol II initiation under uracil-shortage conditions. Because mutation of the upstream TATA box prevents activation of the TSSmRNA, it must be postulated that transcription through the R box is required for internal initiation to occur. The R box and the required T-rich elements might be recognized at the DNA level, and transcription through the region might alter the chromatin structure to allow ex novo polymerase entry. Alternatively, the nascent RNA copy of the R box might function to attract a new polymerase for initiation at TSSCUT. In this case, specific RNA-binding factors might recognize the T-rich motifs of the R box.
(B) In the presence and absence of uracil, a constant level of readthrough occurs at the TSSCUT during the scanning process (dashed arrow). In the presence of uracil, scanning polymerases do not cross the R box or cannot recognize the TSSmRNA and eventually dissociate from the template. When uracil is limiting, scanning through the R box is productive and leads to selection of the downstream TSSmRNA. T-rich sequence features within the R box are required for this to occur, which might be relevant to recognition of these elements by specific factors and/or to the regulated alteration of the nuclosomal structure of the region. In (A) and (B), the TSSCUT and TSSmRNA are indicated, respectively, by black and white boxes. Gray boxes and filled circles indicate the Nrd1 complex binding sites and the T-rich regions within the R box, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions