Can terminators be used as insulators into yeast synthetic gene circuits?
© The Author(s) 2016
Received: 17 October 2016
Accepted: 4 December 2016
Published: 16 December 2016
In bacteria, transcription units can be insulated by placing a terminator in front of a promoter. In this way promoter leakage due to the read-through from an upstream gene or RNA polymerase unspecific binding to the DNA is, in principle, removed. Differently from bacterial terminators, yeast S. cerevisiae terminators contain a hexamer sequence, the efficiency element, that strongly resembles the eukaryotic TATA box i.e. the promoter sequence recognized and bound by RNA polymerase II.
By placing different yeast terminators (natural and synthetic) in front of the CYC1 yeast constitutive promoter stripped of every upstream activating sequences and TATA boxes, we verified that the efficiency element is able to bind RNA polymerase II, hence working as a TATA box. Moreover, terminators put in front of strong and medium-strength constitutive yeast promoters cause a non-negligible decrease in the promoter transcriptional activity.
Our data suggests that RNA polymerase II molecules upon binding the insulator efficiency element interfere with protein expression by competing either with activator proteins at the promoter enhancers or other RNA polymerase II molecules targeting the TATA box. Hence, it seems preferable to avoid the insulation of non-weak promoters when building synthetic gene circuit in yeast S. cerevisiae.
where k lk is the mRNA production rate constant due to promoter leakage, k tr is the transcription initiation rate, T represents a transcription factor that binds p and regulates m synthesis, k d is m decay rate, and c is a coefficient equal to 1 if T is an activator or 0 when T is a repressor. In principle, the leakage term can be omitted if promoter p is repressed since the Hill function equals zero only for an infinite amount of repressor T, whereas it has to be present in case of transcription activation. In this scenario, the leakage is entirely due to RNA polymerase II binding to a promoter in an inactive configuration i.e. either already occupied by repressor proteins or in absence of any activator that can help RNA polymerase II recruitment. However, leakage effects can be due also to RNA polymerase II moving along the DNA after a read-through from an adjacent terminator or an unspecific binding upstream a promoter TATA box–the entry point for RNA Polymerase II along a promoter sequence . Therefore, even constitutive promoters, which can be regarded as always active, can be affected by leakage. Thus, in order to quantify the strength of a constitutive promoter properly, one should insulate the promoter, perhaps with the insertion of a terminator upstream its sequence .
Yeast terminators are characterized by three specific small sequences: the efficiency element, the positioning element, and the poly(A) site . The efficiency element plays an important role in 3’ end formation. Its length and nucleotide composition varies from terminator to terminator. The strongest termination signal corresponds to the efficiency element TATATA . RNA polymerase II can bind a promoter and start transcription in absence of enhancers  and promoter strength is highly dependent on its TATA box . The sequences TATAAA  and TATAAAA  are the strongest TATA boxes in yeast S. cerevisiae and mammalian cells. However, the TATATA hexamer, i.e. the strongest terminator efficiency element, is also reported to be a strong TATA box . Therefore, insulating a promoter with a terminator that contains the efficiency element TATATA corresponds to place a strong TATA box in front of a promoter.
In this work we show that the TATATA and other efficiency elements can indeed work as TATA boxes since they are able to recruit RNA polymerase II to the DNA and lead the transcription of a green fluorescence protein. This was proved with the construction of new synthetic promoters where a native or a synthetic terminator was placed in front of the yeast CYC1 promoter stripped of its two upstream activating sequences (UAS) and its three TATA boxes [12, 13]. As a consequence of their ability to bring RNA polymerase II to the DNA, terminators can disturb the transcriptional activity of a promoter if employed as insulators. All terminators used in this work decreased the expression of a green fluorescent protein when placed in front of the strong yeast GPD promoter . Furthermore, the same terminator (from the DEG1 yeast gene ) showed different degrees of transcription down-regulation when placed in front of promoters of diverse strength. Hence, RNA polymerase II binding the insulator efficiency element interferes with activator proteins binding their enhancers and/or other RNA polymerase molecules that initiate transcription at the promoter TATA box. We show that a short distance between the efficiency element and the promoter UAS decreases protein expression drastically. However, insulator negative effects on transcription are still manifest when the efficiency element and the promoter UAS are considerably far away from each other. We also show that the strength of the efficiency element as a TATA box is another determinant factor of a decrease in protein expression. Moreover, we argue that DNA bending due to strong activators can foster the competition among RNA polymerases and the activator themselves to get access to the DNA. The experimental data illustrated in the next section allows us to point out when, in the construction of yeast synthetic gene circuits, promoter insulation should be avoided and when, in contrast, it is less likely to influence protein synthesis and, therefore, the performance of a whole circuit.
Results and discussion
Building new synthetic promoters by using terminator sequences
List of our new synthetic promoters with full specification of their TATA boxes (nt stands for nucleotides)
Distance from the TSS (nt)
Interestingly, pCYC1noTATA alone gives a fluorescence amount that, although low (18.5 Arbitrary Units–AU), is clearly above both the background (4.3 AU) and the shortADH1t-pCYC1noTATA (5.9 AU) fluorescence level. If we regard shortADH1t-pCYC1noTATA as an insulated pCYC1noTATA, we can conclude that leakage effects due to unspecific binding of RNA polymerase II upstream the pCYC1noTATA sequence account only for few (about 12) arbitrary units of fluorescence (with the machine setup we chose for our measurements). Therefore, they are negligible for strong promoters such as pGPD, whose average fluorescence level is about 612 AU.
Insulating the strong GPD promoter
Transcription units into bacterial synthetic gene circuits can be insulated by placing a terminator in front of the promoter. Bacterial promoters contain, around position −10, a TATA-like hexamer recognized by RNA polymerase. However, bacterial terminators are very different from the eukaryotic ones: they have a palindromic region rich in guanines and cytosines followed by at least six thymines. Upon transcription, the G-C-rich sequence folds into an hairpin that slows down the motion of RNA polymerase. Taking advantage of the weak U−A bond between mRNA and DNA in the active site once the T-rich motif has been transcribed, RNA polymerase can escape from the DNA.
From the results on the terminator-pCYC1noTATA synthetic promoters, we concluded that leakage effects on constitutive strong promoters should be negligible. Therefore, if the four terminators we chose worked as pure insulators with no interference whatsoever with pGPD transcription initiation process, we would expect to see only a small difference between the fluorescence levels of the four terminator-pGPD constructs and the one of the wild-type pGPD. Roughly, a properly-insulated pGPD would express about 98% of the fluorescence measured on the wild-type pGPD.
Insulating different promoters
Structures of the five yeast promoters we insulated with DEG1t
Distance DEG1t(TATAAA)-UAS (nt)
Placing an insulator between two transcription units
Used as an insulator, a terminator should stop RNA polymerase II that bound to an unspecific site upstream the promoter sequence from reaching the promoter TATA box and start mRNA transcription. This leakage effect should be removed to characterize promoter strength properly and, therefore, improve synthetic gene circuit design. However, yeast terminators contain a hexamer, the efficiency element, that is in general very similar to a TATA box. In particular, the strongest efficiency element, TATATA, is identical to one of the strongest eukaryotic TATA boxes.
By fusing four different terminators in front of what we called pCYC1noTATA (i.e. the sequence of the yeast constitutive CYC1 promoter from which we removed all the UASes and TATA boxes) we proved that the efficiency elements are able to recruit RNA polymerase II molecules from the cell nucleus and lead to fluorescent protein expression. Moreover, we could estimate that the contribution of leakage effects to constitutive promoter transcriptional activity is very low.
When we placed the same terminators in front of the strongest yeast constitutive promoter, pGPD, fluorescence production dropped remarkably. This reduction could not be explained by the sole leakage removal. Hence, we concluded that the terminator efficiency elements interferes with transcription initiation by recruiting RNA polymerase II molecules.
Among the four terminators we used in this work, DEG1t proved to be the one with higher affinity towards RNA polymerase II. We insulated five yeast constitutive promoters of different strength with the DEG1 terminator. Only the weakest among these promoters (pACT1) turned out to be insensitive to the insulator presence, the other four underwent a non negligible reduction in transcription efficiency. In particular, ADH1 promoter fluorescence level dropped to the 7% of its original value. This effect was due to the short distance between the DEG1t efficiency element and pADH1 UAS (43 nucleotides only). RNA polymerase II binding the insulator efficiency element is, however, able to interfere with the promoter transcriptional activity also when a high distance separates the efficiency element from the promoter UAS. This was apparent from our experiments on a re-engineered GPD promoter where the strong bipartite UAS was almost completely mutated such that transcription was activated by a weaker UAS downstream. Once insulated with DEG1t, the fluorescence level of the mutated pGPD dropped by about one half, despite the fact that the distance between the efficiency element and the weak UAS was higher than 450 nucleotides.
Probably, DNA bending is a factor that enhances the interaction between RNA polymerase II binding the insulator efficiency element and either the activators targeting the enhancers or other RNA polymerase II binding the promoter TATA box. We argue that, by integrating two adjacent transcription units into the same locus of the S. cerevisiae genome, the effect of DNA bending is somehow lowered. This would explain why DEG1t, once inserted between two transcription units, caused only a marginal reduction in the expression of the downstream protein.
Overall, our results point out clearly that a single transcription unit containing a strong or medium-strength promoter should not be insulated with a terminator when integrated into the genome of yeast S. cerevisiae. Insulation, indeed, would provoke a reduction in protein expression that might have high repercussions on the performance of a whole synthetic gene circuit.
The yeast integrative shuttle-vector plasmid pRSII406 (Addgene-35442, a gift from Steven Haase)  was used as a backbone for the construction of every transcription unit. pGPD, pACT1, the CYC1 promoter (pCYC1), and the genomic CYC1 terminator (genCYC1t) were extracted from the yeast S. cerevisiae genome (strain FY1679-08A, see below) following the procedure in . pCYC1 served as a template to PCR out the sequence of pCYC1noTATA and pCYC1min. pTEF2 was obtained from the MIT Registry part BBa_K801010, pTEF1 was extracted via PCR from pRS404-pTEF1-Ago1 (Addgene-22313, a gift from David Bartel), pADH1 was obtained from pHCA/GAL4(1-93).ER.VP16  (courtesy of Picard lab, University of Geneva, Switzerland).
Every transcription unit expresses either the yomKate2 red fluorescent protein obtained from pFA6a-link-yomKate2-CaURA3 (Addgene-44878, a gift from Wendell Lim and Kurt Thorn) or the yeast enhanced green fluorescent protein (yEGFP) obtained from pRS31-glag  (courtesy of Hasty lab, University of California, San Diego, USA). A slightly different sequence (yEGFPgg) was used in the plasmids constructed with the MoClo method . Here, the internal BsaI site was removed via silent mutation. The CYC1 terminator (CYC1t), placed at the end of every transcription unit, is described in  and is slightly different from genCYC1t . We obtained it from pRS403-pGAL1-strongSC_GFP (Addgene-22316, a gift from David Bartel).
The ADH1 terminator used in this work (shortADH1t) was constructed by using, as a template, the MIT Registry part BBa_K801012. Insulated promoters were extended via PCR to be preceded by DEG1t (50 nt long), Tsynth8 (49 nt long) or their mutated version. The spacer s100 was taken from the bacterial tetR gene. The constructs genCYC1t-pCYC1noTATA, Tsynth8-s100-pCYC1noTATA, DEG1-pCYC1noTATA (for the MoClo assembly, see Additional file 1), and part of mut_pGPD were synthesized by GENEWIZ Inc. (Suzhou, China).
Plasmids were constructed either via isothermal assembly  or MoClo method . For the MoClo assembly, we used the original universal 0-level acceptor vector pAGM9121 (Addgene-51833, a gift from Sylvestre Marillonnet)  and the 1-level acceptor vector ypL1F-1_406 that we constructed by adapting pRSII406 to host a transcription unit between the BpiI cutting sites TGCC and GCAA (for the original pL1F-1 see ). Primers for PCR were designed according to the chosen DNA assembly method.
Touchdown PCR was employed to select and amplify DNA sequences. DNA elution from agarose gel was carried out with the QIAGEN-28604 “DNA Elution kit”. Isothermal assembly required always one hour at 50 °C. In order to assemble 1-module plasmids the insert (0-modules) and our ypL1F-1_406 1-level acceptor vector were combined in 2:1 molar ratio and mixed with a master mix (1 μ l BsaI 20 units /μ l, NEB-R0535S; 2 μ l Cutsmart buffer NEB; 1 μ l T4 ligase 400 units /μ l, NEB-M0202S; 2 μ l10 m M ATP, Sigma-Aldrich-A7699) to a final 15 μ l volume. The thermocycler program was set to: 3 cycles of 10 min at 40 °C and other 10 min at 16 °C. These cycles were followed by 10 min at 50 °C, 20 min at at 80 °C, and the final temperature was set to 16 °C. E. coli competent cells (strain DH5 α, Life Technology 18263-012) transformed with our plasmids (30-s heatshock at 42 °C) were grown overnight at 37 °C either in LB broth or plates (Bacto-tryptone 10%, Yeast extract 5%, NaCl 10%, Agar 15% for the plates) supplied with the necessary antibiotic (ampicillin or spectinomycin). Plates were spread with 100μ l100 m M IPTG (Merck) and 100μ l20 m g/m l X-gal for blue/white screening. Plasmid extraction from bacterial cells was carried out with standard methods . All the plasmids were further sequenced (Sanger method) to check the correctness of the newly assembled synthetic constructs.
Yeast strain construction
Our new, synthetic plasmids were integrated into the genome of the yeast S. cerevisiae strain FY1679-08A (MATa; ura3-52; leu2 Δ1; trp1 Δ63; his3 Δ200; GAL2), Euroscarf (Johann Wolfgang Goethe University, Frankfurt, Germany). Genomic integration was carried out as described in . About 5 μ g of plasmidic DNA were linearized either along the URA3 marker with the restriction enzyme StuI (NEB-R0187S) or along the LEU2 marker with the restriction enzyme BstXI (NEB-R0113S). Transformed cells were grown on plates containing synthetic selective medium (SD-URA or SD-LEU; 2% glucose, 2% agar) for about 36 h at 30 °C.
Yeast cells were grown overnight in synthetic complete medium (SDC) at 30 °C. They were diluted, in the morning, approximately 1:100 and let them grow (in synthetic medium again) up to five more hours such that their O D 600 was always between 0.2 and 2.0 (exponential phase). Fluorescence measurements were performed with a BD FACScalibur machine (488 n m laser, 530/30 filter). The FACS machine set-up was reproduced at each experiment by using fluorescent beads (AlignFlow, Life Technologies-A16500). We placed their peak (mean value) as close as possible to 400 AU. The measurement was repeated at the end of each experiment to assure that the machine conditions did not change considerably over the whole experiment. We considered as reliable only the measurements where the relative difference between the initial and the final value of the peaks of the beads was lower than 5%. Data were analyzed with the flowcore R-Bioconductor package . Statistically significant difference between two fluorescence levels was estimated via two-sided Welch’s t-test (p-value <0.05). Fluorescence levels were estimated as the mean values of at least three independent experiments (i.e. carried out in different days–each time 30000 samples were recorded). Standard deviations were calculated on these mean values. The error on a relative fluorescence value (ratio) was finally computed via the error propagation formula. Box plots and histograms of representative experiments are provided in the Additional file 1.
We want to thank Prof Jeff Hasty for kindly providing us with the plasmid pRS31-glag and Prof Didier Picard for kindly providing us with the plasmid pHCA/GAL4(1-93).ER.VP16. We also want to thank the other students of the Synthetic Biology lab together with Prof Ying Hu and her students, and Mr Yao Zhang for their help in various lab tasks and useful discussions. We are also grateful to Ms Lei Yue for her assistance with FACS experiments.
We acknowledge financial support by the National Natural Science Foundation of China grant number 3157080528.
Availability of data and material
A complete list of the plasmids and the yeast strains we constructed, together with the DNA sequences of each genetic part (promoter, CDS, and terminator) used in this work are provided in the Additional file 1. Further experimental data and model results, which support our conclusions, are included in the Additional file 1 as well.
Main project idea: MAM. Plasmid construction and integration into yeast cells: WS, JL, and LQ. FACS experiments and data analysis: WS and MAM. Manuscript writing: MAM. All authors read and approved the final manuscript.
The authors declare they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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