Nucleic Acids Research

Nucleic Acids Res. 37(18): e120-e120

A fast and efficient translational control system for conditional expression of yeast genes

Peter Kötter¹², Julia E. Weigand¹³, Britta Meyer¹², Karl-Dieter Entian¹²*, Beatrix Suess¹³*

¹Institut für Molekulare Biowissenschaften, ²Cluster of Excellence: Macromolecular Complexes, Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M., Germany and ³Aventis Foundation Endowed Professorship

*To whom correspondence should be addressed. Tel: +49 69 798 29785; Fax: +49 69 798 29323; Email: suess@bio.uni-frankfurt.de

Correspondence may also be addressed to Karl-Dieter Entian. Tel: +49 69 798 29525; Fax: +49 69 798 29323; Email: entian@bio.uni-frankfurt.de

© 2009 The Author(s)

DOI: 10.1093/nar/gkp578

Published in print: October 2009

Published online: 10 July 2009

Abstract

A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5′UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression.

INTRODUCTION

Although the yeast model organism Saccharomyces cerevisiae was the first eukaryote to have its genome sequenced (1), the molecular and physiological functions of many of the encoded proteins are still not understood; especially difficult to investigate are essential genes. Since more than one-third of the essential S. cerevisiae genes have homologous human counterparts, yeast has become an important model organism for genome functional analysis in eukaryotes. For the investigation of essential gene functions, conditional gene expression is an indispensable tool and several such systems have been developed which regulate either transcription or protein stability (2).

For transcriptional gene regulation natural yeast promoters, such as the GAL1 and the MET25 promoters are used. Target genes are expressed either in the presence of galactose (3) or the absence of methionine (4). However, such regulation always interferes with cellular metabolism due to the changes in growth media composition and in many cases regulation is not tight enough to completely prevent growth. A major improvement was the introduction of heterologous tc-regulated promoters which are either inducible or repressible (TetON/OFF) (5–7). A clear advantage of these systems is that tc does not severely interfere with yeast cellular metabolism (8) and nowadays, these strains are commercially available (9).

Alternatively, the stability of essential proteins can be targeted to achieve conditional expression. Most suitable for gene function analyses are temperature-sensitive mutants with the respective proteins loosing their function upon a shift to non-permissible temperatures, which are either increased (temperature-sensitive ts mutants) or decreased (cold-sensitive cs mutants). Unfortunately, ts and cs mutants are isolated in specific strain backgrounds and, thus, cannot be easily transferred to non-isogenic strains. The fusion of heat-inducible degron sequences to essential proteins combined with their expression by the copper-inducible CUP1 promoter makes the respective target protein proteolytically sensitive upon temperature shift. This provides an alternative method for ts-regulated gene expression (10,11).

Despite many successful applications of the above mentioned systems, an easy adaptable regulatory system for conditional gene expression in yeast is still missing. We developed a new concept for conditional yeast gene expression which regulates translation of the target mRNAs by inserting a tc aptamer with strong binding affinity for tc (12) into the 5′UTRs. In contrast to protein-based systems, such aptamer-based synthetic riboswitches rely on a direct RNA–ligand interaction, are thus strain independent and no interference of heterologous expressed regulatory proteins is expected. Because of its small size and its regulatory properties, the aptamer is perfectly suited to control expression of any gene of interest. Here, we describe an easy adaptable PCR-based system, which exploits the principal of direct RNA–ligand interaction for the efficient conditional control of essential genes in yeast.

MATERIALS AND METHODS

Plasmid constructions and genetic manipulations

Established protocols for molecular biology were followed (13). Yeast strains were transformed with the lithium acetate procedure (14).

pADH1-(tc)n-GFP

We used a 108-nt-long 5′UTR from pWHE702 (15) which originates from the pTEF promoter and carries an additional 70-nt-long CAA spacer to adjust its length to a 5′UTR which contains one copy of an aptamer. This 108-nt-long fragment was cloned into pWHE602 in front of a pADH1-driven GFP reporter gene (16) resulting in pADH1-GFP. The sequence of the complete 5′UTR with the CAA spacer and all restriction sites is given in the Supplementary Table S1. Overlap-PCR was used to insert one, two and three aptamers, resulting in plasmids pADH1-tc1-GFP, pADH1-tc2-GFP or pADH1-tc3-GFP, respectively (Figure 1A). Sequences for all aptamer-containing 5′UTRs are included in Supplementary Table S1.

Figure 1.View larger versionFigure 1. Tc aptamer-mediated translational inhibition. (A) Schematic view of the aptamer-containing mRNAs. The cap structure is shown as a ball. (B) Secondary structure of the tc aptamer [stem = pedestal (P), bulge (B) and loop (L)]. B1-2 and L3 are important for tc binding as it is supported by structural probing (34) and the crystal structure (35).

pADH1-tc3

The ADH1 promoter and the tc aptamer containing 5′UTR were PCR amplified from pADH1-tc3-GFP. The HA-tag was amplified from the vector pOM10 (17) (kindly provided by EUROSCARF, www.uni-frankfurt.de/fb15/mikro/EUROSCARF) and attached via overlap-PCR. The PCR products were introduced into pUG6 (18) using the two unique restriction sites SalI and BsiWI next to a kanamycin-resistance gene flanked by loxP sites resulting in pADH1-tc3.

pTDH3-(tc)n

The tc aptamer containing 5′UTRs were PCR amplified from pADH1-(tc)n-GFP. The HA-tag was amplified from the vector pOM10 (17) and attached via overlap-PCR. The PCR product was introduced in pPK421 (19) via the unique restriction site BsiWI which is downstream of the transcription initiation site of the TDH3 promoter and next to a kanamycin-resistance gene flanked by loxP sites resulting in pTDH3-tc1, -tc2 or -tc3, respectively.

All plasmids, maps and complete sequences are available from EUROSCARF (http://www.uni-frankfurt.de/fb15/mikro/EUROSCARF/index.html).

See full table

See full table

GFP measurements

S. cerevisiae strain RS453α transformed with the respective constructs was grown at 28°C for 48 h in 5 ml of minimal medium [0.2% (w/v) yeast nitrogen base, 0.55% ammonium sulfate, 2% (w/v) glucose, 12 µg/ml adenine, MEM amino acids, Gibco BRL] in the absence or presence of 250 µM tc. Cells were harvested by centrifugation and resuspended in 2 ml phosphate-buffered saline (PBS). For each construct, three independently grown cultures were analyzed. Fluorescence measurements were carried out at 25°C on a Fluorolog FL3-22 (Horiba Jobin Yvon) with the excitation wavelength set to 482 nm and an emission wavelength of 510 nm. Optical density (OD₆₀₀) was determined to ensure homogeneous cell growth. The vector pVT102-U (20) without the GFP gene was analyzed in parallel as a blank and its value was subtracted from all data.

Genomic integration of the aptamer-containing integration cassettes

All genetic modifications of the S. cerevisiae genome were performed via homologous recombination using PCR products with 40-nt-long flanking homologies to the target genes (21). The insertion cassettes were PCR amplified from the corresponding vectors pADH1-tc3 and pTDH3-(tc)n using the primer pairs x-Tc1 and x-Tc2. Primer pairs x-Tc1B and x-Tc2 can be used for amplification of the integration cassettes without HA-tag. Sequences of all primers used for the amplification of the integration cassettes are given in Supplementary Table S2. After transformation of S. cerevisiae strain CEN.PK122 (22) with the PCR products, correct integration in G418 resistant strains was confirmed by PCR using primer pairs x-A1/K2 and x-A2/ADH1-A5 and TDH3-A7, respectively (promoter-specific primer, see Figure 2B and Supplementary Table S2) and by sequencing. Diploid transformants were subsequently analyzed by tetrad dissection.

Figure 2.View larger versionFigure 2. Universal insertion cassettes for tc aptamer-mediated regulation. (A) Schematic representation of the insertion cassette with a kanamycin-resistance gene (KAN; kanMX4 modul) flanked by loxP sites. The promoters pADH1 or pTDH3 are fused to a synthetic 5′UTR with n copies of the tc aptamer (n = 1, 2 and 3, respectively) followed by an HA-tag (either 3xHA or 6xHA) for protein quantification. All plasmid-encoded insertion cassettes can be PCR amplified with a single pair of target gene specific primers (x-Tc1 and x-Tc2). x-Tc2 shares 40-nt homology with the promoter and x-Tc1 40-nt homology with the open reading frame of the target gene. The double line represents the target chromosomal locus. (B) Chromosomal situation after integration. The gene of interest is expressed by a heterologous promoter (either pADH1 or pTDH3) and controlled by n copies of the tc aptamer in its 5′UTR. Primers to control successful integration are indicated.

Serial dilution growth assay

Yeast cells were grown overnight in 1% yeast extract, 2% peptone supplemented with 2% glucose (YPD) or 2% fructose (YPF) to an OD₆₀₀ of 1-2. Cells were diluted in normal saline to an OD₆₀₀ of 1 followed by 3-fold 1:10 serial dilution. From the diluted cultures, 5 µl were spotted onto YPD plates in the absence (control) and presence of tc. Growth differences were recorded following incubation of the plates for 2–3 days at 30°C.

Western blot analysis

Protein extracts were prepared from HA-epitope tagged strains using glass beads. Total protein concentration was determined with the micro-biuret method (23,24). Equivalent amounts of protein were separated with 10% SDS polyacrylamide gels and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore) by electrotransfer. Membranes were blocked with 5% nonfat dry milk and HA-tagged proteins were detected with 1:5000 dilutions of the 12CA5 anti-HA monoclonal antibody (Roche), followed by a second reaction with a 1:30 000 dilution of anti-mouse IgG-conjugated horseradish peroxidase (BioRad) for the use with ECL chemoluminescence protocol (GE Healthcare).

Northern blot analysis

RNA was prepared as previously described (25); 18.4 µg total RNA was separated on 1% agarose gels (in 1xTAE) supplemented with 6.66% formaldehyde and transferred to a positively charged nylon membrane (Hybond N+, GE Healthcare) via capillary blotting. 10 pmol oligonucleotides NB_HA (hybridizes to the HA-tag) and NB_ACT1_1 (hybridizes to ACT1-mRNA) were radioactive labeled at the 5′ end using 6 µl γ-³²P-ATP (∼3.3 pmol/µl, Hartmann-Analytik) and 1 µl T4 polynucleotide kinase (Roche) in the supplied buffer for 1 h at 37°C and purified with Illustra Microspin G-25 columns (GE Healthcare); 23 µl-labeled oligonucleotides (∼600.000 c.p.m/µl) were used as probe for one membrane. Hybridization was done in 12 ml RotiHybriQuick (Roth) overnight at 42°C. Signals were visualized by phosphoimaging using a Typhoon 9100 (GE Healthcare).

RESULTS

The tc aptamer has been used to control gene expression on different levels, including translation initiation (16,26) and pre-mRNA splicing (27). The use of two tc aptamer-regulated introns or combination of translational and splicing control significantly increased efficiency of regulation (27). Therefore, we questioned if the insertion of multiple copies of the tc aptamer into the 5′UTR also increases regulation.

As shown for an ADH1 promoter-driven GFP reporter gene, one tc aptamer (pADH1-tc1-GFP) reduced basal GFP expression to ∼28% and constructs with two (pADH1-tc2-GFP) or three tc aptamers (pADH1-tc3-GFP) had 24 and 21% residual activity, respectively (Table 1). In all cases, addition of tc resulted in a further strong reduction of GFP fluorescence with 8-fold for a construct with one, 21-fold with two and 37-fold with three tc aptamer copies, respectively.

See full tableTable 1. Regulatory properties of tc aptamer insertion

To test the potential of tc aptamer inhibition for conditional regulation of essential genes in yeast we developed an easy, adaptable and flexible PCR-based strategy for genomic tagging. Homologous flanking regions were used for all constructs to integrate different promoters with tc aptamer-containing 5′UTRs directly in front of the ATG start codons of the selected open reading frames, thereby uncoupling them from their endogenous promoters and 5′UTRs. This allows the use of only a single pair of gene-specific PCR primers to amplify all available promoter-aptamer constructs (Figure 2). To compensate for the decreased translation efficiency, the glycolytic TDH3 and ADH1 promoters with different expression strengths were used. The insertion cassette also harbours an HA-tag for detection of the proteins by western blots and a kanamycin resistance gene flanked by loxP sites, as selection marker for chromosomal integration. Five essential genes (NEP1, NOP8, NOP14, PGI1 and SEC1) were chosen as proof of concept for tc-dependent translational regulation. For two of them, NOP8 and SEC1, attempts to establish conditional regulation using other systems had failed so far (8,28).

Transformation of wild-type yeast with PCR amplified insertion cassettes containing three tc aptamers led to colony formation on medium containing G418. The correct insertion of the pTDH3-tc3- and pADH1-tc3-cassettes in front of all five genes was confirmed by an analytic PCR of genomic DNA and by sequencing. This shows that the PCR-based strategy is easily adaptable and efficient enough to target any gene within the yeast genome.

In the absence of tc, all five constructs driven by the TDH3 promoter fully complemented the endogenous promoters and 5′UTRs. For NEP1, SEC1, NOP8 and NOP14, the TDH3-tc3 constructs provided normal growth to the respective insertion strains. In contrast, after tetrad analysis of heterozygous asci, the pTDH3-tc3-PGI1 constructs showed reduced growth when compared to wild-type segregants (Figure 3A). For the highly expressed PGI1 gene, pTDH3-tc3 promoter strength was obviously not sufficient to permit normal growth in glucose-containing media. However, with fructose as carbon source growth of TDH3-tc3-PGI1 segregants was like wild type (data not shown). Expression mediated by the less efficient pADH1-tc3 promoter was not sufficient to complement the substitution of the endogenous NEP1 and PGI1 promoters (Figure 3A).

Figure 3.View larger versionFigure 3. Tc aptamer-regulated expression of essential genes. (A) Segregation pattern of tc aptamer-regulated genes after tetrad analysis. Diploid strain CEN.PK122 was transformed with the tc aptamer insertion cassettes resulting in a heterozygous strain carrying a wild type and a tc aptamer modified allele of the target gene. After meiosis, the viability of the tc-regulated target genes was followed by tetrad dissection. For the pTDH3-driven constructs, four viable segregants were obtained for all target genes (upper four lanes). No viable segregants were obtained for the pADH1-driven PGI1 and NEP1 (2:2 segregation). (B) Growth dilution assays were performed for wild-type and insertion mutants. Cultures were grown overnight in YPD medium and diluted to a final density of OD₆₀₀ of 1.0 (1). Ten-fold serial dilutions (corresponding to 2, 3 and 4) were spotted on YPD medium with increasing concentrations of tc. Colonies were grown for three days at 30°C. (C) Regulation of a pTDH3-driven NEP1 gene with one (pTDH3-tc1-NEP1), two (pTDH3-tc2-NEP1) or three (pTDH3-tc3-NEP1) copies of the tc aptamer.

Serial dilution growth assays showed that increasing concentrations of tc (Figure 3B) abolished growth of all pTDH3-tc3-constructs. However, while 100 µM tc was sufficient to prevent growth of the pTDH3-tc3-NEP1 and pTDH3-tc3-PGI1 strains, 500 µM tc were needed to abolish growth of the pTDH3-tc3-NOP8 and pTDH3-tc3-SEC1 strains. The less active ADH1 promoter was more suitable for controlling NOP8 (pADH1-tc3-NOP8) and SEC1 (pADH1-tc3-SEC1) expression where 100 µM tc completely prevented growth.

Taken together, our data clearly show that both the expression strength of the promoter and the tc concentration influence the efficiency of regulation. This allows the system to be easily adjusted to the individual expression level of the target gene.

The number of tc aptamers inserted also contributed to tc-dependent translational control. As exemplified for NEP1, no regulation was achieved with only one aptamer present in the 5′UTR, whereas addition of a second tc aptamer provided efficient regulation. Although a third tc aptamer copy further improved the strength of regulation in the GFP system (Table 1), no difference with respect to growth regulation was observed for constructs with two or three copies (Figure 3C).

The efficiency of tc aptamer inhibition was shown for pTDH3-tc3-NOP14 which encodes an essential protein needed for ribosome biogenesis. Addition of tc to liquid medium results in a reduced growth rate within 4 h. Furthermore, Nop14 protein levels decreased to <50% 2 h after tc addition and almost completely disappeared within 6 h (Figure 4).

Figure 4.View larger versionFigure 4. Repression kinetic of tc aptamer-controlled NOP14 expression. (A) Growth curves of a strain with NOP14 regulated by the tc aptamer (pTHD3-tc3-NOP14). A culture was diluted to an OD₆₀₀ of 0.1 in YPD media and incubated for 2.5 h at 30°C. After splitting, one aliquot was treated with 200 µM tc. Growth was followed for 8 h (black dots, no tc, gray dots, 200 µM tc). (B) Samples were taken at indicated time points after treatment with tc and Nop14-HA protein amounts determined by western blot analysis.

Northern blot analyses of the TDH3-tc3-NOP14 strains showed that the NOP14 mRNA was stable for at least 6 h after tc addition (Figure 5A). This indicates that the TDH3-tc3-NOP14 gene was further transcribed after tc addition and that the tc-bound mRNA was not degraded. Remarkably, the ACT1 mRNA signal, which was used as an internal control, strongly decreased after 6 h in the TDH3-tc3-NOP14 strain. This degradation is probably the result of a diminished number of ribosomes due to the Nop14-mediated arrest of ribosome biogenesis. For the TDH3-tc3-PGI1 gene, which encodes the glycolytic phosphoglucose isomerase enzyme, PGI1 and ACT1 mRNAs stayed stable (Figure 5B).

Figure 5.View larger versionFigure 5. Northern blot analysis of tc aptamer-controlled NOP14 and PGI1 expression. Strains with (A) pTDH3-tc3-3HA-NOP14 and (B) pTDH3-tc3-3HA-PGI1 were grown in YPD media (NOP14) and YPF (PGI1), respectively. Overnight cultures were grown to an OD₆₀₀ of 0.4 at 30°C. After splitting, one aliquot was treated with 200 µM tc. Samples were taken at indicated time points after treatment with tc and total RNA was prepared. mRNA amounts of NOP14 and PGI1 were determined by northern blot analysis. Actin mRNA (ACT1) was used as internal control.

Taken together, our results impressively show that tc aptamer-induced inhibition causes an immediate translational response upon tc addition.

DISCUSSION

Conditional regulation is an indispensable tool for the functional analysis of essential genes. Most commonly target gene expression is addressed at the transcriptional level. However, not all essential genes can be controlled by transcriptional regulation and in some cases these systems give false-positive results due to enhancing or silencing effects, such as the recently reported trans activation (29). This study, therefore, introduces an alternative regulatory system that targets translation. Previously, we have shown that the ligand-bound form of the tc aptamer when inserted into the 5′UTR inhibits binding of the small ribosomal subunit to the cap structure and the formation of the 80S ribosome without any effect on the mRNA steady state level (15). In contrast, insertion of the aptamer into the 3′UTR leads to no regulation (unpublished data). For all five cases studied here, conditional translational regulation was successfully achieved, including two genes for which conditional regulation at the level of transcription had failed so far.

The simple and efficient technique using PCR to tag genomic loci with tc-dependent aptamers allows fast and efficient regulation of any yeast gene of interest. The promoter strength is of major importance for successful conditional regulation. Here, we provide several insertion cassettes, which allow adjustment to the individual expression level of the target gene via a combination of two different promoters and the number of tc aptamers. For easy handling all integration cassettes can be amplified with a single pair of target specific primers.

The striking advantage of our system is that no further auxiliary protein factors are necessary so that no interference of heterologous expressed regulatory proteins is expected. Furthermore, the application is completely strain independent. The effector tc is pharmacologically well characterized, with good cell permeability and without any influence on mRNA stability, protein expression and growth (8,15). Our experiments with NOP14 have shown that protein synthesis is switched off immediately upon tc addition. The relative amount of Nop14p decreased by 50% after one generation time. This inversely corresponds to the 2-fold increase in overall protein amount after one cell cycle.

Our work greatly expands the applicability of aptamer-controlled gene expression systems. For the first time we demonstrate the control of essential, endogenous genes by an RNA aptamer-based regulation system. This is possible since the system is tight and independent of positioning effects. Until now only three examples for endogenous (but not essential) genes regulated by aptamers have been reported. A synthetic riboswitch, which responds to theophylline engineered by the Gallivan group, shows robust increase in gene expression upon theophylline addition. However, this system is restricted to gene regulation in bacteria since regulation involves sequestration of the bacterial ribosomal binding site (30,31). Conditional gene expression by RNAi controlled by aptamers has also been shown (32,33). However a specific design of the regulating elements is needed for each new gene. So far, our system is applicable in S. cerevisiae and possibly other yeasts. Attempts to regulate genes in human cell lines and plants failed so far probably due to stronger helicase activities (unpublished data).

Taken together, we established a tight tc-dependent translational control system, which allows regulated expression of yeast genes with one single PCR step. Therefore, it is a highly suitable method to study the physiological function of essential and nonessential yeast proteins.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

FUNDING

The Aventis Foundation; the Volkswagenstiftung (I/79 950); the Deutsche Forschungsgemeinschaft (SU 402/1-2, SFB579); the Cluster of Excellence: Macromolecular Complexes; and the Goethe-University. Funding for open access charge: Deutsche Forschungsgemeinschaft (DFG).

Conflict of interest statement. None declared.

Supplementary Material

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ACKNOWLEDGEMENTS

The authors wish to thank C. Berens for critically reading the manuscript. We thank S. Ballweg, S. Lamberts and P. Watzinger for excellent technical assistance.

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

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Figure 1.

Tc aptamer-mediated translational inhibition. (A) Schematic view of the aptamer-containing mRNAs. The cap structure is shown as a ball. (B) Secondary structure of the tc aptamer [stem = pedestal (P), bulge (B) and loop (L)]. B1-2 and L3 are important for tc binding as it is supported by structural probing (34) and the crystal structure (35).

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Acc.-No | Plasmid
p30595 | pADH1-tc1-GFP
p30596 | pADH1-tc2-GFP
p30597 | pADH1-tc3-GFP
p30598 | pADH1-tc3-3xHA
p30599 | pADH1-tc3-6xHA
p30600 | pTDH3-tc3-3xHA
p30601 | pTDH3-tc3-6xHA
p30602 | pTDH3-tc2-3xHA
p30603 | pTDH3-tc2-6xHA
p30604 | pTDH3-tc1-3xHA
p30605 | pTDH3-tc1-6xHA

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Strains
CEN.PK122 | MATa/MATα MAL2-8c/MAL2-8c SUC2/SUC2 (9)
RS453α | MATα ade2-1 trp1-1 can1-100 leu2-3 his3-1 ura3-52

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Figure 2.

Universal insertion cassettes for tc aptamer-mediated regulation. (A) Schematic representation of the insertion cassette with a kanamycin-resistance gene (KAN; kanMX4 modul) flanked by loxP sites. The promoters pADH1 or pTDH3 are fused to a synthetic 5′UTR with n copies of the tc aptamer (n = 1, 2 and 3, respectively) followed by an HA-tag (either 3xHA or 6xHA) for protein quantification. All plasmid-encoded insertion cassettes can be PCR amplified with a single pair of target gene specific primers (x-Tc1 and x-Tc2). x-Tc2 shares 40-nt homology with the promoter and x-Tc1 40-nt homology with the open reading frame of the target gene. The double line represents the target chromosomal locus. (B) Chromosomal situation after integration. The gene of interest is expressed by a heterologous promoter (either pADH1 or pTDH3) and controlled by n copies of the tc aptamer in its 5′UTR. Primers to control successful integration are indicated.

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Table 1.

Regulatory properties of tc aptamer insertion

Construct | Relative fluorescence (%) no tca | Relative fluorescence (%) 250 µM tca | Regulatory factorb
pADH1-tc1-GFP | 28.0 | 3.6 | 8
pADH1-tc2-GFP | 23.8 | 1.1 | 21
pADH1-tc3-GFP | 20.5 | 0.6 | 37

aGfp fluorescence expressed by the vector without an aptamer (pADH1-GFP, Figure 1A) was set to 100% and corresponds to 1.1 × 10⁶ counts per second (cps) without and 1.3 × 10⁶ cps with 250 µM tc, respectively, with background level subtracted (8 × 10³ cps without and 16 × 10³ cps with 250 µM tc, respectively); values are mean of three independently grown cultures with standard deviation below 7%.

bEfficiency of regulation is given as the ratio of respective values with and without tc.

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Figure 3.

Tc aptamer-regulated expression of essential genes. (A) Segregation pattern of tc aptamer-regulated genes after tetrad analysis. Diploid strain CEN.PK122 was transformed with the tc aptamer insertion cassettes resulting in a heterozygous strain carrying a wild type and a tc aptamer modified allele of the target gene. After meiosis, the viability of the tc-regulated target genes was followed by tetrad dissection. For the pTDH3-driven constructs, four viable segregants were obtained for all target genes (upper four lanes). No viable segregants were obtained for the pADH1-driven PGI1 and NEP1 (2:2 segregation). (B) Growth dilution assays were performed for wild-type and insertion mutants. Cultures were grown overnight in YPD medium and diluted to a final density of OD₆₀₀ of 1.0 (1). Ten-fold serial dilutions (corresponding to 2, 3 and 4) were spotted on YPD medium with increasing concentrations of tc. Colonies were grown for three days at 30°C. (C) Regulation of a pTDH3-driven NEP1 gene with one (pTDH3-tc1-NEP1), two (pTDH3-tc2-NEP1) or three (pTDH3-tc3-NEP1) copies of the tc aptamer.

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Figure 4.

Repression kinetic of tc aptamer-controlled NOP14 expression. (A) Growth curves of a strain with NOP14 regulated by the tc aptamer (pTHD3-tc3-NOP14). A culture was diluted to an OD₆₀₀ of 0.1 in YPD media and incubated for 2.5 h at 30°C. After splitting, one aliquot was treated with 200 µM tc. Growth was followed for 8 h (black dots, no tc, gray dots, 200 µM tc). (B) Samples were taken at indicated time points after treatment with tc and Nop14-HA protein amounts determined by western blot analysis.

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Figure 5.

Northern blot analysis of tc aptamer-controlled NOP14 and PGI1 expression. Strains with (A) pTDH3-tc3-3HA-NOP14 and (B) pTDH3-tc3-3HA-PGI1 were grown in YPD media (NOP14) and YPF (PGI1), respectively. Overnight cultures were grown to an OD₆₀₀ of 0.4 at 30°C. After splitting, one aliquot was treated with 200 µM tc. Samples were taken at indicated time points after treatment with tc and total RNA was prepared. mRNA amounts of NOP14 and PGI1 were determined by northern blot analysis. Actin mRNA (ACT1) was used as internal control.

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