1. Regulation of a Eukaryotic Gene by GTP-Dependent Start Site Selection and Transcription Attenuation 
Jason N. Kuehner, David A. Brow 
Molecular Cell 
Volume 31, Issue 2, Pages (July 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1 Model of Regulation of IMD2 Expression by GTP
IMD2 encodes IMPDH, which converts IMP to XMP, a precursor of GMP. Imd2 mRNA level correlates inversely with guanine nucleotide level. Use of TATA-proximal G start sites when GTP levels are high results in synthesis of noncoding attenuated transcripts that are rapidly degraded by the nuclear exosome. Use of a TATA-distal A start site when GTP levels are low bypasses the attenuator and produces Imd2 mRNA. The drugs 6-azauracil (6-AU) and mycophenolic acid (MPA) inhibit IMPDH, reducing intracellular GTP pools and inducing IMD2 expression (Exinger and Lacroute, 1992; Jenks and Reines, 2005). Numbers indicate distance in base pairs from the mRNA start site. The box indicates the IMD2 open reading frame. The expected location of the Sen1-dependent terminator (−55 to +42) is based on mapping of a repressive element (Shaw et al., 2001; Escobar-Henriques et al., 2003; Kopcewicz et al., 2007).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Factor Dependence of Attenuation at NRD1, HRP1, and IMD2
(A) Schematic diagram of the NRD1-, HRP1-, and IMD2-CUP1 reporter genes.
(B) Copper resistance assay for attenuator function. Ten-fold serial dilutions of cup1Δ strains, that are otherwise wild-type (WT) or contain a sen1-E1597K, nrd1-5, nab3-11, ssu72-G33A, hrp1-5, or rpb11-E108G mutation and harbor NRD1-CUP1, HRP1-CUP1, or IMD2-CUP1 reporter plasmids, were spotted onto medium lacking uracil with the indicated concentration of copper sulfate and with or without MPA (10 μg/mL). Strains were grown at 25°C for 7 days. The top row shows the cup1Δ strain harboring a CUP1 plasmid as a positive control.
(C) Northern blot analysis of Hrp1 mRNA. RNA from the PSY1 (WT) or PSY1076 (hrp1-5) strains grown at permissive temperature (25°) or shifted for 2 hr to restrictive temperature (37°C) was probed for Hrp1 mRNA and scR1 (SRP RNA), which serves as a loading control in this and subsequent figures.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Cis-Acting Mutations that Disrupt IMD2 Regulation
(A) Point mutations that induce expression of the IMD2-CUP1 reporter gene in the absence of MPA are shown by arrows below the nucleotide residue. Numbers indicate position in base pairs from the IMD2 mRNA start site. The TATA box and CUP1 start codon are boxed. Bent arrows indicate transcription start sites. The locations of introduced EcoRI sites are underlined. The T-71A mutation, which alters a start codon to allow proper translation of readthrough transcripts, is indicated by inverse font. The two major polyadenylation sites for attenuated transcripts are indicated by asterisks.
(B) Deletion mutations that disrupt IMD2 regulation in the context of the IMD2-CUP1 reporter gene.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Mutations in the IMD2 Promoter and 5′UTR Alter Transcription Start Site Selection and Recognition of the Attenuator, Disrupting Regulation by GTP
(A) Copper resistance assay for IMD2 attenuator function. Ten-fold serial dilutions of cultures of a cup1Δ strain harboring CUP1, IMD2-CUP1, or the indicated mutant IMD2-CUP1 reporter plasmids were spotted onto medium lacking uracil, with or without added copper sulfate. Strains were grown at 30°C for 3 days.
(B) Northern analysis of Cup1 mRNA. RNA from cup1Δ strains harboring wild-type or mutant IMD2-CUP1 reporter plasmids grown in the absence or presence of MPA was probed for Cup1 mRNA and scR1.
(C) Primer extension analysis of RNA preparations shown in (B). Lanes 7 and 8 contain the products of sequencing reactions with the same primer on wild-type IMD2-CUP1 DNA.
(D) Primer extension analysis of Cup1 mRNA from cis- and trans-acting mutants that confer copper resistance.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
GTP Sensitivity of Upstream Initiation Depends on the Identity of +1 and +2 Initiating Nucleotides
(A) Schematic diagram of the IMD2-CUP1 Δ−102 to +106 reporter gene. The attenuator and +1A site are deleted from this allele, leaving upstream G starts to drive initiation.
(B) Mutant alleles of the −141G, −126G, and −116G start sites at positions +1 and +2. Substitutions are indicated by inverse font.
(C) Copper resistance assay for G start site function. Ten-fold serial dilutions of a cup1Δ strain harboring IMD2-CUP1 and wild-type or mutant IMD2-CUP1 Δ−102 to +106 reporter plasmids were spotted onto medium lacking uracil, with or without added copper sulfate, with or without added guanine (250 μM), and with or without MPA (15 μg/mL). Strains were grown at 30° for 3 days.
(D) Primer extension analysis of Cup1 mRNA from the IMD2-CUP1 Δ−102 to +106 reporter strain grown under conditions of high or low GTP. The location of G or A start sites and the junction between IMD2 and CUP1 sequences are indicated.
(E) Averaged quantitation of three independent sets of RNA preparations and primer extension experiments, under conditions shown in (D). Total TSS is the sum of signal from all start sites shown in (D). Error bars show standard error.
(F) Primer extension analysis of Cup1 mRNA from wild-type or mutant IMD2-CUP1 Δ−102 to +106 reporter strains.
(G) Quantitation of primer extension data shown in (F).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Mutations that Decrease the GTP Dependence of Initiation at the Upstream Starts of IMD2
(A) Mutations in the IMD2-CUP1 Δ−102 to +106 allele that confer copper resistance, with single nucleotide substitutions and deletions (Δ) shown below the wild-type sequence. The underlined sequence is tandemly duplicated in the Dup −194 to −158 mutant. The TATA box, an EcoRI site introduced with two mutations (bold), and the CUP1 start codon are boxed.
(B) Copper resistance assay for “G” start site function. Ten-fold serial dilutions of cup1Δ strains harboring wild-type or mutant IMD2-CUP1 Δ−102 to +106 reporter plasmids were spotted onto medium lacking uracil with added copper sulfate and with or without MPA (15 μg/mL). Strains were grown at 30° for 3 days.
(C) Primer extension of RNA from the indicated mutant strains grown in the presence of MPA.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 GTP-Dependent Regulation of Endogenous IMD2 and PSA1 Genes
(A) Northern blot analysis of Imd2 mRNA from RPB1, rpb1-D260N, and rpb1-N488D strains grown in the presence or absence of MPA.
(B) Primer extension analysis of Imd2 mRNA from RPB1, rpb1-D260N, and rpb1-D260N strains grown in the presence or absence of added guanine for 2 hr at 30°C.
(C) Primer extension analysis of Imd2 mRNA from SUA7 (TFIIB gene), sua7-W63P, sua7-R64A, and sua7-F66D strains.
(D) Primer extension analysis of Psa1 mRNA from PSA1 and PSA1-G-149A strains grown in the presence or absence of MPA. A sequencing ladder was run on the same gel. U4 RNA serves as a loading control.
(E) Models for negative or positive regulation of Pol II transcription by [iGTP]. The G starts are GTP-concentration sensitive, and “term” indicates a Sen1-dependent terminator.
Molecular Cell  , DOI: ( /j.molcel )
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