- Open Access
Evaluation of GFP reporter utility for analysis of transcriptional slippage during gene expression
© The Author(s) 2018
- Received: 19 June 2018
- Accepted: 17 September 2018
- Published: 21 September 2018
Epimutations arising from transcriptional slippage seem to have more important role in regulating gene expression than earlier though. Since the level and the fidelity of transcription primarily determine the overall efficiency of gene expression, all factors contributing to their decrease should be identified and optimized.
To examine the influence of A/T homopolymeric sequences on introduction of erroneous nucleotides by slippage mechanism green fluorescence protein (GFP) reporter was chosen. The in- or out-of-frame gfp gene was fused to upstream fragment with variable number of adenine or thymine stretches resulting in several hybrid GFP proteins with diverse amino acids at N-terminus. Here, by using T7 phage expression system we showed that the intensity of GFP fluorescence mainly depends on the number of the retained natural amino acids. While the lack of serine (S2) residue results in negligible effects, the lack of serine and lysine (S2K3) contributed to a significant reduction in fluorescence by 2.7-fold for polyA-based in-frame controls and twofold for polyTs. What is more, N-terminal tails amino acid composition was rather of secondary importance, since the whole-cell fluorescence differed in a range of 9–18% between corresponding polyA- and polyT-based constructs.
Here we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP hybrid fluorescence mainly depends on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. Moreover, only in case of GFP hybrids with relatively short N-terminal tags the fluorescence level solely reflects production yield, what further indicates the impact of an individual slippage sequence. Our results demonstrate that in contrast to the E. coli enzyme, T7 RNA polymerase exhibits extremely high propensity to slippage even on runs as short as 3 adenine or 4 thymine residues.
- Green fluorescent protein reporter
- Transcriptional slippage
- A/T homopolymers
- T7 RNA polymerase
- E. coli RNA polymerase
Green fluorescent protein (GFP) from the Aequorea victoria jellyfish  is nowadays the most widely used and developed reporter applicable in biochemistry and cell biology . GFP and its mutant derivatives possess internal fluorophore that re-emits light which covers visible spectrum of colors . Hence, GFP technology is very attractive and easily adaptable to biotechnology and many fields of basic science. GFP is used as a protein fusion reporter in various applications in diverse kind of cells for imaging and detection of very specific processes, such as fusion gene expression, tagged protein in situ location, host–parasite interactions and others . The hybrid protein maintains its normal function along with fluorescent property acquired from GFP expressing gene. The incorporation of GFP can be done at either the N- or C-terminus of a protein or peptide domain.
Epigenetic effects of ribosomal and transcriptional frameshifting play important role in regulating gene expression processes as it has clearly emerged from the accumulated and still growing knowledge in this field . Our research concerns comparative analysis of the transcriptional slippage propensity of the two most widely used RNA polymerases (RNAPs): E. coli and T7 bacteriophage, representing two different families of enzymes . Long mononucleotide A/T stretches destabilize and realign RNA:DNA hybrid, contributing to RNAP “slippage” throughout the DNA template. Productive transcriptional slippage at such sites involves unwinding-rewinding of the RNA:DNA hybrid which is not sensed by RNAP active site and hereby does not lead to enzyme backtracking and mRNA correction. Forward and backward mRNA slippage generates insertion/deletion errors in nascent transcripts, resulting in shift of reading frame . It is known that the error rate of transcripts generated by E. coli RNAP in vivo is significantly high, roughly between the values of 10−5 and 10−3 per residue , and even higher for T7 RNAP . Insertion/deletion (indels) type of transcriptional errors may be considered as ambiguous. From one side they are detrimental to efficiency of gene expression, but on the other hand can also be beneficial for cell physiology. Transcriptional slippage has significant potential to restore the wild-type phenotype of indel mutant genes [6, 8–13]. Here, we evaluated GFP protein reporter for potential use in a study of the transcriptional slippage phenomenon which occurs during expression of out of frame fusion genes with upstream fragments containing A/T homopolymeric sequences. The results demonstrate utility of GFP fusion gene to study transcriptional slippage effect in homopolymer sequence-dependent manner.
Bacterial strains and culture conditions
All cloning was performed using Escherichia coli (E. coli) DH10B strain (F− λ− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara leu) 7697 galU galK rpsL nupG) grown at 37 °C in Luria broth (LB)  supplemented with 50 μg/ml kanamycin or 100 μg/ml ampicillin for maintenance of pET24a- or pBAD24-based plasmids, respectively [15, 16]. For fluorescence experiments E. coli ER2566 T7 phage RNAP IPTG inducible strain (F− λ− fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11 Δ(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2 R(zgb-210::Tn10-TetS) endA1 [dcm]) was used (New England Biolabs, Ipswich, USA).
Standard protocols  and kits were used for purification of plasmid DNA (A&A Biotechnology, Gdynia, Poland), digestion of DNA with restriction endonucleases, ligation of DNA with T4 DNA ligase and PCR techniques with PfuPlus DNA polymerase (all from EURx, Gdansk, Poland).
pET24a derivative reporter vectors (Additional file 1: Table S1) were constructed using PCR to amplify a 900-bp fragment of pGreenTIR plasmid , a source of enhanced fluorescence gfp variant (F64L/S65T) , and different set of primers (Additional file 1: Table S2) provided three reading frames (− 1, 0 and + 1). The resulting PCR fragment containing the promoterless gfp gene was digested with BamHI and EcoRI and cloned into the same sites of pET24a. Next, sets of two annealed oligonucleotides (Additional file 1: Table S2) carrying appropriate polyA/T sequences were inserted into NheI–BamHI sites and ligated. All resulting fusion genes were confirmed by sequencing (Genomed, Warszawa, Poland), and then, as XbaI–HindIII fragments were subcloned into pBAD24 plasmid.
Site-specific mutagenesis using PCR was carried by high fidelity PfuPlus DNA polymerase (Eurx) according to the manufacturer’s instructions (50 ng of plasmid template was added to a 50-μl PCR reaction). Appropriate plasmid templates and nucleotide deletion/insertion in reverse primers were PCR-amplified and parental plasmid was eliminated by digestion with DpnI enzyme (10 u, Thermo Scientific, Waltham, USA) for 1.5-h at 37 °C. Products were resolved in agarose gels, appropriate bands were cut out and aliquots containing purified DNA were transformed into DH10B competent cells. All plasmid modifications were confirmed by Sanger DNA sequencing using the BigDye Terminator v3.1 (Applied Biosystems, Waltham, USA) (Genomed). Additional file 1: Tables S1 and S2 include a list of plasmids and primers used.
Whole cell fluorescence
ER2566 cells were grown in LB medium, containing kanamycin or ampicillin, at 37 °C, until the culture reached an OD600 of 0.2–0.3. Then, to induce expression of tested fusion genes isopropyl β-d-1-thiogalactopyranoside (IPTG) or l-arabinose were added to 1 mM or 0.1%, respectively, and cells were further incubated for additional 1 h. Cells were gently harvested (400 μl sample), resuspended in 200 µl F buffer (M9 salts; 0.1 mM CaCl2; 1 mM MgSO4) and then quantified using a Varioskan® Flash Spectral Scanning Multimode Reader (Thermo Scientific) at excitation and emission wavelengths of 485 and 510 nm, respectively. The raw fluorescence intensity of each culture was normalized to cell density (OD600) and the background fluorescence from gfp-less cells was subtracted from each reading.
Western blotting for GFP protein
Culture extracts, after normalization to OD600, were analyzed by 10% SDS-PAGE, transferred into nitrocellulose membrane and GFP protein detection was performed using mouse monoclonal anti-GFP (B-2) antibodies (Santa Cruz Biotechnology, Dallas, USA) diluted 1:4000 in TBS-T buffer (50 mM Tris–HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.6) with 5% skimmed milk for 1.5 h at room temperature. After three washes with TBS-T, the membrane was incubated for 1 h with chicken anti-mouse IgG-HRP (horseradish peroxidase, 1:5000, Santa Cruz Biotechnology). The membrane was washed three times and the specific protein was visualized by adding chemiluminescent substrate solution (Pierce ECL Plus Western Blotting Substrate, Thermo Scientific) and exposed to X-ray film. For M2.MboII detection membrane was probed with rabbit anti-M2.MboII antibodies  diluted 1:1250 in TBS-T buffer, followed by incubation with a goat anti-rabbit secondary antibody conjugated with alkaline phosphatase (1:30,000, Sigma-Aldrich, Saint Louis, USA). A specific protein was visualized by adding BCIP/NBT substrate solution (Thermo Scientific).
RNA extraction and cDNA synthesis
The cells were proceed as described in Whole cell fluorescence except after 1 h incubation with appropriate inducer cellular RNA was extracted with Total RNA Mini Plus Concentrator Kit (A&A Biotechnology) according to the manufacturer’s instructions. For the mRNA stability experiment culture samples were taken starting 30 s prior to addition of rifampicin (250 μg/ml, BioShop, Burlington, Canada). Culture sample volumes were corrected for OD to maintain similar cell numbers per sample. Samples were immediately mixed with 0.5 ml of stayRNA protection buffer (A&A Biotechnology). cDNA were obtained after RNase-free DNase I (Thermo Scientific) treatment by using RevertAid First Strand cDNA Synthesis Kit (Thermo Scietific).
Northern blot detection
Profiles of the gfp fusion genes specific transcripts were analyzed by northern blotting. Equal amounts (5 μg) of total RNA were loaded on formaldehyde denaturing 1.3% agarose gel and then transferred onto Zeta-Probe® blotting membrane (BioRad, Hercules, USA) by capillary forces. PCR-produced 774 bp dsDNA fragment specific to the whole gfp gene sequence (obtained with primers bamGFP6 and Gfpdown, Additional file 1: Table S2, and pGreenTIR as a template) after biotin labelling (Biotin-High Prime, Roche Diagnostics, Basel, Switzerland) was used as a probe. Chemiluminescent detection was carried using streptavidin-HRP Pierce ECL substrate (Thermo Scientific) and exposure to X-ray film.
The specific primers, designed to ensure similar Tm and PCR product size, are given in Additional file 1: Table S2. 16S rrn was chosen as internal stable reference housekeeping gene, while in case of gfp fusion variants 5′-end of transcripts was analyzed. Real-time PCR with LightCycler 2.0 (Roche Diagnostics) was performed in triplicate in three independent experiments using SG qPCR mix with SYBR Green (Eurx). The PCR employed the following cycling parameters: 95 °C for 5 min, followed by 35 cycles of 94 °C for 20 s, 60 °C for 20 s, 72 °C for 10 s each; and finally the melting curve (60–97 °C) program for quality control, and cooling to 40 °C. The levels of remaining mRNA for each variant was normalized to the level of the reference housekeeping gene 16S rrn, and then determined by fitting the percentage of mRNA remaining vs. time to an exponential decay function. The relative fold-change mRNAs ratios were obtained by normalizing each time point data in reference to the earliest measurements. In all studies the half-lives of transcripts were determined by fitting the percentage of mRNA remaining vs. time to an exponential decay function. The relative fold-change mRNAs ratios were obtained by normalizing each time point data in reference to the earliest measurements .
Analysis of the N-terminal amino acid sequence of the A5GFP-1 hybrid
The N-terminal protein sequence analysis was performed at BioCentrum Ltd. (Krakow, Poland). Sequentially detached phenylthiohydantoin derivatives of amino acids were identified using the Procise 491 (Applied Biosystems) automatic sequence analysis system, according to the standard protocol of the manufacturer.
Contribution of a short and long N-terminal tags to the GFP fluorescence ability
Identification of GFP reporter slippage product
Testing the proximal part of gfp gene for the possibility of an “surplus” slippage by T7 RNAP
To make sure that the detectable GFP fluorescence is owed only to the tested slippage sequence, the proximal part of gfp gene was examined for generating unexpected slippage or translational initiation from an alternative start codons. We checked possibility of production of truncated but functional GFP protein, by constructing − 1 frameshifted gfp-1 fusion without any long slippery sequence (pETmingfp-1) and found it to manifest low fluorescence level, little above background (Fig. 2a, b). We reasoned that slight GFP protein production/activity was possible through slippage occurred at alternative sites located in the proximal part of gfp: AAA (27–29 nt) or TTTT (40–43 nt) (Fig. 2b, c). Such amazing ability of T7 RNAP to slip on the template containing at least three nucleotide repeats was already reported in in vitro experiments . We confirmed possibility of bypassing single nucleotide deletion through slippage at T4 site by construction a pETmin(T5)gfp0 vector with in-frame gfp fusion gene “hopping” into the appropriate 0 frame by addition of a single T to the normally present four (15th amino acid). The resulting mutant variant GFP[T5]0, despite lacking 7 native N-end amino acids (M1A2S3L4A5P6P7D8P9K10E11K12N13F14F15(8)T16(9)G17(10)) exhibited 22% fluorescence compared to GFPA60 positive control. Moreover, the importance of T4 run in restoration of the proper frame was confirmed by interruption of its continuity (gfp[TGT2]-1). Such a construct (pETmin(TGT2)gfp-1) showed decreased fluorescence by about 27% compared to pETminA2gfp-1 with full T4 run, yet its level was 4 times higher than background fluorescence (Fig. 2b, c). This suggested likely contribution of AAA sequence in slippage induction, what was further verified with a pETmin(A2G)gfp-1construct in which triple A sequence was modified (gfp[A2G]-1). Indeed, we observed subsequent decrease in GFP activity/production, but it reached little over the background.
To exclude the possibility, that fluorescent active GFP protein might be produced from a rare alternative start codon ATT , pETgfpATT140 was constructed. In this variant gfp gene began from the 14th codon, which in the wild type is ATT, here substituted with ATG. Although we observed production of hybrid protein (Fig. 2d), yet no fluorescence was detected (Fig. 2b). This result is in agreement with data obtained by Raghunathan . Interestingly, we detected remnant production of GFP even in case of − 1 frameshifted gfpNull-1 and gfpNhe-1 genes which lacked any triple A/T nucleotide in the 5′-terminal part of fusion gene (Fig. 2d). All of this indicates extremely high inclination of T7 RNAP to erroneous transcription, quite opposite to E. coli host RNAP (Fig. 2d).
Relation between the intensity of hybrid GFP fluorescence and the number of retained natural amino acids
GFP reference normalization requirements
In this work, we present experimental evidence for utility of GFP reporter for accurate estimation of A/T homopolymeric sequence contribution in transcriptional slippage induction. We showed that the intensity of GFP fluorescence mainly depended on the number of retained natural amino acids, thus fluorescence raw data need to be referred to appropriate positive control. This GFP reporter-based tool can be easily applied to study of any slippage sequence.
MS and EW conceived and designed the research; EW, DK, MSz and MS performed the experiments, analyzed the data. EW and MS wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
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This work was supported by the National Science Centre (Poland) (2012/07/BNZ2/01782 to M.S.).
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