Activity Of T7 RNA Polymerase Research Articles

RT-IVT method allows multiplex real-time quantification of in vitro transcriptional mRNA production

For the past 30 years, in vitro transcription (IVT) technology has been extensively used for RNA production or for basic transcriptional mechanism research. However, methods for mRNA quantification still need to be improved. In this study, we designed a RT-IVT method using binary fluorescence quencher (BFQ) probes and the PBCV-1 DNA ligase to quantify mRNA production in real-time by fluorescence resonance energy transfer (FRET) and RNA-splinted DNA ligation. Compared with existing methods, the RT-IVT method is inexpensive and non-radioactive, and can detect mRNA production in unpurified systems in real-time and shows high sensitivity and selectivity. The activity of T7 RNA polymerase and Escherichia coli RNA polymerase holoenzyme was then characterized with this method. We then multiplexed the real-time mRNA quantification for three T7 promoters on a RT-PCR thermocycler by using BFQ probes with different colored fluorophores that were specific for each target. Ultimately, we created an inexpensive multiplexed method to quantify mRNA production in real-time, and future research could use these methods to measure the affinity of transcriptional repressors to their target DNA sequence.

An Assay Method for Characterizing Bacteriophage T7 RNA Polymerase Activity by Transcription-Translation (TX-TL) System.

T7 system is a commonly used in protein expression and the highest transcription activity of T7 RNAP usually caused the instability of T7 system. In order to apply T7 system extensively, it is essential to characterize T7 RNAP activity. In the present paper, an assay method for T7 RNAP activity was developed with a transcription-translation (TX-TL) system. After the optimization of TX-TL system, the operating parameters were determined as 34°C, 60min with 20ng/μl of plasmid DNA template. The standard curve of TX-TL assay method indicated an excellent correlation (r = 0.998), and the sensitivity was better than that of western blotting method. The precision investigation indicated a mean-relative error of 2.58% and a standard-relative error of 7.01%. Moreover, the cell lysate could be added directly to the optimized TX-TL system without affecting T7 RNAP activity assay. The feasibility of present method was further confirmed by characterizing T7 RNAP activity in cell lysate of five strains of Escherichia coli (E. coli) DH5α with different T7 RNAP activities and seven commercial strains of E. coli (DE3). The present assay method for T7 RNAP activity would have a great application in synthetic biology, metabolic engineering, enzyme engineering and biomedicine.

Plasmids for Controlled and Tunable High-Level Expression in E. coli.

Controlled gene expression is crucial for engineering bacteria for basic and applied research. Inducible systems enable tight regulation of expression, wherein a small-molecule inducer causes the transcription factor to activate or repress transcriptional initiation. The T7 expression system is one of the most widely used inducible systems, particularly for high overexpression of proteins. However, it is well known that the highly active T7 RNA polymerase (RNAP) has several drawbacks, including toxicity to the host and substantial leaky expression in the absence of an inducer. Much work has been done to address these issues; current solutions require special strains or additional plasmids, making the system more complicated and less accessible. Here, we challenge the assumption that the T7 expression system is the best choice for obtaining high protein titers. We hypothesized that expression from strong inducible promoters expressed from high-copy plasmids could compete with expression levels obtained from T7 RNAP but that such promoters would possess improved control of transcription. Employing inducible systems from a toolbox we developed previously, we demonstrate that our plasmids consistently give higher outputs and greater fold changes over basal expression than the T7 system across rich and minimal media. In addition, we show that they outperformed the T7 system when we used an engineered metabolic pathway to produce lycopene. IMPORTANCE Genetic systems for protein overexpression are required tools in microbiological and biochemical research. Ideally, these systems include standardized genetic parts with predictable behavior, enabling the construction of stable expression systems in the host organism. Modularity of a genetic system is advantageous, so that the expression system can be easily moved into a host that best suits the needs of a given experiment. The T7 expression system lacks both predictability and stability and requires special host strains to function. Despite these limitations, it remains one of the most popular systems for protein overproduction. This study directly compared the T7 system to four inducible systems from our broad-host-range plasmid toolbox and demonstrated these alternative expression systems have distinct advantages over the T7. The systems are entirely plasmid-based and not constrained to a specific bacterial host, expanding the options for high-level protein expression across strains.

High-throughput iSpinach fluorescent aptamer-based real-time monitoring of in vitro transcription

In vitro transcription (IVT) is an essential technique for RNA synthesis. Methods for the accurate and rapid screening of IVT conditions will facilitate RNA polymerase engineering, promoter optimization, and screening for new transcription inhibitor drugs. However, traditional polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography methods are labor intensive, time consuming and not compatible with real-time analysis. Here, we developed an inexpensive, high-throughput, and real-time detection method for the monitoring of in vitro RNA synthesis called iSpinach aptamer-based monitoring of Transcription Activity in Real-time (STAR). STAR has a detection speed at least 100 times faster than conventional PAGE method and provides comparable results in the analysis of in vitro RNA synthesis reactions. It also can be used as an easy and quantitative method to detect the catalytic activity of T7 RNA polymerase. To further demonstrate the utility of STAR, it was applied to optimize the initially transcribed region of the green fluorescent protein gene and the 3T4T variants demonstrated significantly enhanced transcription output, with at least 1.7-fold and 2.8-fold greater output than the wild-type DNA template and common transcription template, respectively. STAR may provide a valuable tool for many biotechnical applications related to the transcription process, which may pave the way for the development of better RNA-related enzymes and new drugs.Graphical iSpinach aptamer-based monitoring of transcription activity in real-time(STAR).

Dual Promoters Improve the Rescue of Recombinant Measles Virus in Human Cells.

Reverse genetics is a technology that allows the production of a virus from its complementary DNA (cDNA). It is a powerful tool for analyzing viral genes, the development of novel vaccines, and gene delivery vectors. The standard reverse genetics protocols are laborious, time-consuming, and inefficient for negative-strand RNA viruses. A new reverse genetics platform was established, which increases the recovery efficiency of the measles virus (MV) in human 293-3-46 cells. The novel features compared with the standard system involving 293-3-46 cells comprise (a) dual promoters containing the RNA polymerase II promoter (CMV) and the bacteriophage T7 promoter placed in uni-direction on the same plasmid to enhance RNA transcription; (b) three G nucleotides added just after the T7 promoter to increase the T7 RNA polymerase activity; and (c) two ribozymes, the hairpin hammerhead ribozyme (HHRz), and the hepatitis delta virus ribozyme (HDVrz), were used to cleavage the exact termini of the antigenome RNA. Full-length antigenome cDNA of MV of the wild type IC323 strain or the vaccine AIK-C strain was inserted into the plasmid backbone. Both virus strains were easily rescued from their respective cloned cDNA. The rescue efficiency increased up to 80% compared with the use of the standard T7 rescue system. We assume that this system might be helpful in the rescue of other human mononegavirales.

A single mutation attenuates both the transcription termination and RNA-dependent RNA polymerase activity of T7 RNA polymerase

ABSTRACT Transcription termination is one of the least understood processes of gene expression. As the prototype model for transcription studies, the single-subunit T7 RNA polymerase (RNAP) is known to respond to two types of termination signals, but the mechanism underlying such termination, especially the specific elements of the polymerase involved, is still unclear, due to a lack of knowledge with respect to the structure of the termination complex. Here we applied phage-assisted continuous evolution to obtain variants of T7 RNAP that can bypass the typical class I T7 terminator with stem-loop structure. Through in vivo selection and in vitro characterization, we discovered a single mutation (S43Y) that significantly decreased the termination efficiency of T7 RNAP at all transcription terminators tested. Coincidently, the S43Y mutation almost eliminates the RNA-dependent RNAP (RdRp) activity of T7 RNAP without impeding the major DNA-dependent RNAP (DdRp) activity of the enzyme. S43 is located in a hinge region and regulates the transformation between transcription initiation and elongation of T7 RNAP. Steady-state kinetics analysis and an RNA binding assay indicate that the S43Y mutation increases the transcription efficiency while weakening RNA binding of the enzyme. As an enzymatic reagent for in vitro transcription, the T7 RNAP S43Y mutant reduces the undesired termination in run-off RNA synthesis and produces RNA with higher terminal homogeneity.

Technical challenges in the development of reverse genetics for a viral haemorrhagic septicaemia virus (VHSV) genotype Ib isolate: Alternative cell lines and general troubleshooting

Several reverse genetics systems for viral haemorrhagic septicaemia virus (VHSV) have been developed over the last decade. These systems have been based on genotype Ia, IVa and IVb isolates and have used the fish cell line EPC, which is less susceptible to some VHSV isolates belonging to genotype I and genotypes II and III. While developing a reverse genetics system in our laboratories for VHSV genotype Ib, we realized that the isolate in interest (SE SVA 1033 9C) did not grow in EPC cells and it was necessary to adapt the reverse genetics protocols to the BF-2 fish cell line. This cell line is very sensitive to high temperatures and is therefore not compatible with the original protocols based on the use of recombinant vaccinia virus (vTF7-3) as a provider of the T7 RNA polymerase (T7-RNAP) to the system, which includes incubation periods at 37 °C. Transfection efficiency was assessed in BF-2 cells using a reporter plasmid and it showed to be highest when using Lipofectamine™ 3000 compared to other transfection reagents. A luciferase assay was performed to determine the optimal activity of T7-RNAP in BF-2 cells with different amounts of vTF7-3. We successfully recovered recombinant VHSV (rVHSV) in BF-2 cells by reducing the incubation time at 37 °C after transfection to both 3 and 6 h. Another strategy we attempted successfully was to transfect mammalian BHK-21 cells, which are routinely used to propagate vTF7-3, and after the 37 °C incubation period, a BF-2 cell suspension was added hypothesizing that the virions formed in the transfected mammalian cells would infect the subsequently added fish cells at 15 °C incubation over the following days. We have successfully recovered rVHSV from both BHK-21 with a BF-2 cells suspension as well as a new protocol for VHSV reverse genetics in BF-2 cells has been established.

Development of High‐Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches

AbstractNucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine‐ and deoxycytidine‐derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high‐performance photoswitches with near‐quantitative, fully reversible back‐and‐forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4‐fold turn‐off factor, demonstrating the potential for optochemical control of biological processes.

Development of High-Performance Pyrimidine Nucleoside and Oligonucleotide Diarylethene Photoswitches.

Nucleosidic and oligonucleotidic diarylethenes (DAEs) are an emerging class of photochromes with high application potential. However, their further development is hampered by the poor understanding of how the chemical structure modulates the photochromic properties. Here we synthesized 26 systematically varied deoxyuridine‐ and deoxycytidine‐derived DAEs and analyzed reaction quantum yields, composition of the photostationary states, thermal and photochemical stability, and reversibility. This analysis identified two high‐performance photoswitches with near‐quantitative, fully reversible back‐and‐forth switching and no detectable thermal or photochemical deterioration. When incorporated into an oligonucleotide with the sequence of a promotor, the nucleotides maintained their photochromism and allowed the modulation of the transcription activity of T7 RNA polymerase with an up to 2.4‐fold turn‐off factor, demonstrating the potential for optochemical control of biological processes.

Toehold‐Mediated Strand Displacement in a Triplex Forming Nucleic Acid Clamp for Reversible Regulation of Polymerase Activity and Protein Expression

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleic acids, target affinity and biological stability superior to that of natural DNA may be obtained. This work demonstrates how a chimeric clamp consisting of acyclic (L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formation of a highly stable triplex structure. The (L)-aTNA clamp is released from the target again by the addition of a releasing strand in a strand displacement type of reaction. It is shown that the clamp efficiently inhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully applied to the regulation of luciferase expression by reversible binding to the mRNA. When targeting a sequence in the double stranded plasmid, 40 % downregulation of protein expression is achieved.

Amplification of RNA for Microarrays.

Gene expression profiling typically requires microgram quantities of mRNA, which can be difficult to obtain. In such cases, RNA must be amplified to have enough material for microarray labeling and hybridization. This protocol generates amplified antisense RNA (aRNA) from limited quantities of total RNA. It is designed around maximizing yield and product length while minimizing template-independent side reactions. Template-independent reactions compete with the desired template-dependent reaction, an undesirable situation that becomes more severe as less RNA template is used. Amplification products dominated by template-independent product result in greatly reduced sensitivity and compression of differences in microarray hybridization experiments. Most notably, the oligo(dT) primer used in reverse transcription (RT) yields a high-molecular-weight product in the in vitro transcription (IVT) reaction independent of any cDNA template. This protocol therefore limits the amount of primer used by using small cDNA synthesis volumes. In addition, high-molecular-weight, template-independent product is generated in the presence of biotinylated NTPs and the absence of any polymer when excessive amounts of T7 RNA polymerase activity are used. Template-dependent product of questionable molecular weight and limited functionality in downstream reactions can also be produced with excessive T7 RNA polymerase activity. Essentially, more yield is not always better. Here, a single round of amplification typically results in a fivefold to 20-fold mass conversion of starting material. If the first-round aRNA is used as a template for a second round of amplification, 200- to 400-fold amplification is typical.

A specific, promoter-independent activity of T7 RNA polymerase suggests a general model for DNA/RNA editing in single subunit RNA Polymerases

Insertional RNA editing has been observed and characterized in mitochondria of myxomycetes. The single subunit mitochondrial RNA polymerase adds nontemplated nucleotides co-transcriptionally to produce functional tRNA, rRNA and mRNAs with full genetic information. Addition of nontemplated nucleotides to the 3′ ends of RNAs have been observed in polymerases related to the mitochondrial RNA polymerase. This activity has been observed with T7 RNA polymerase (T7 RNAP), the well characterized prototype of the single subunit polymerases, as a nonspecific addition of nucleotides to the 3′ end of T7 RNAP transcripts in vitro. Here we show that this novel activity is an editing activity that can add specific ribonucleotides to 3′ ends of RNA or DNA when oligonucleotides, able to form intramolecular or intermolecular hairpin loops with recessed 3′ ends, are added to T7 RNA polymerase in the presence of at least one ribonucleotide triphosphate. Specific ribonucleotides are added to the recessed 3′ ends through Watson-Crick base pairing with the non-base paired nucleotide adjacent to the 3′ end. Optimization of this activity is obtained through alteration of the lengths of the 5′-extension, hairpin loop, and hairpin duplex. These properties define a T7 RNAP activity different from either transcriptional elongation or initiation.

Programmed hierarchical patterning of bacterial populations

Modern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. Here we report a synthetic genetic system implementing population-based AND-logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations.

Engineered Photoactivatable Genetic Switches Based on the Bacterium Phage T7 RNA Polymerase.

Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on properties are obtained with the light-activatable VVD domain and its variants as regulatory domains. For the best split position found, working switches exploit either the light-induced interactions between the VVD domains or allosteric effects. The split fragments show high modularity when they are combined with different regulatory domains such as those with chemically inducible interaction, enabling chemically controlled switches. To summarize, the T7 RNA polymerase-based switches are powerful tools to implement light-activated gene expression in different contexts. Moreover, results about the studied split positions and domain organizations may facilitate future engineering studies on this and on related proteins.

Improving the soluble expression and purification of recombinant human stem cell factor (SCF) in endotoxin-free Escherichia coli by disulfide shuffling with persulfide

We here present a new method for the expression and purification of recombinant human stem cell factor (rhSCF164) in endotoxin-free ClearColi® BL21(DE3) cells harboring codon-optimized Profinity eXact™-tagged hSCF cDNA. Previously, we demonstrated that co-expression with thioredoxin increased the solubility of rhSCF in Escherichia coli BL21(DE3), and addition of l-arginine enhanced chromatography performance by removing the endotoxin-masked surface of rhSCF. Initially, we tried to express rhSCF in an endotoxin-free strain using a thioredoxin co-expression system, which resulted in significantly lower expression, possibly due to the stress imposed by overexpressed thioredoxin or antibiotics susceptibility. Therefore, we developed a new expression system without thioredoxin. External redox coupling was tested using persulfides such as glutathione persulfide or cysteine persulfide for the in vivo-folding of hSCF in the cytoplasm. Persulfides improved the protein solubility by accelerating disulfide-exchange reactions for incorrectdisulfides during folding in E. coli. Furthermore, the persulfides enhanced the expression level, likely due to upregulation of the enzymatic activity of T7 RNA polymerase. The recombinant protein was purified via affinity chromatography followed by cleavage with sodium fluoride, resulting in complete proteolytic removal of the N-terminal tag. The endotoxin-free fusion protein from ClearColi® BL21(DE3) could bind to the resin in the standard protocol using sodium phosphate (pH 7.2). Furthermore, purified rhSCF enhanced the proliferation and maturation of the human mast cell line LAD2. Thus, we conclude that use of the protein expression system employing E. coli by disulfide shuffling with persulfide addition could be a very useful method for efficient protein production.

Construction of a Minigenome Rescue System for Measles Virus, AIK-c Strain

Background:In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus (nucleocapsid protein) N, (phosphoprotein) P and (Large) L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease. Objectives:Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA. Materials and Methods:For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus (CMV) promoter and measles virus N and P proteins were under control of IRES (internal ribosome entry site) sequences. Results:Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line. Conclusions:Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter.

Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control system of plasmid pMV158

Antisense RNAII is a replication control element encoded by promiscuous plasmid pMV158. RNAII binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene. In order to initiate the biochemical characterization of the pMV158 antisense RNA-mediated control system, conditions for in vitro transcription by T7RNA polymerase were set up that yielded large amounts of antisense and target run-off products able to bind to each other. The run-off antisense transcript was expected, and confirmed, to span the entire RNAII as synthesized by the bacterial RNA polymerase, including the intrinsic transcription terminator at its 3′-terminus. On the other hand, two different target transcripts, mRNA60 and mRNA80, were produced, characterized and tested for efficient binding to the antisense product. The mRNA60 and mRNA80 run-off transcripts supposedly spanned 60 and 80 nucleotides, respectively, on the copG-repB mRNA and lacked terminator-like structures at their 3′-termini. Probing of the sequence and conformation of the main products, along with modeling of their secondary structures, showed that both target transcripts were actually longer-than-expected, and contained a 3′-terminal hairpin wherein the extra nucleotides base-paired to the expected 3′-terminus of the corresponding run-off transcript. These longer products were proposed to arise from the RNA-dependent polymerizing activity of T7RNA polymerase on correct run-off transcripts primed by extremely short 3′-selfcomplementarity. Seizing of the target mRNA sequence complementary to the 5′-terminus of RNAII in a stable 3′-terminal hairpin generated by this activity seemed to cause a 3-fold decrease in the efficiency of binding to the antisense RNA.

A Novel Complex: A Quantum Dot Conjugated to an Active T7RNA Polymerase

To perform single-molecule studies of the T7RNA polymerase, it is crucial to visualize an individual T7RNA polymerase, for example, through a fluorescent signal. We present a novel complex combining two different molecular functions, an active T7RNA polymerase and a highly luminescent nanoparticle, a quantum dot. The complex has the advantage of both constituents: the complex can traffic along DNA and simultaneously be visualized, both at the ensemble and at the single-molecule level. The labeling was mediated through anin vivobiotinylation of a His-tagged T7RNA polymerase and subsequent binding of a streptavidin-coated quantum dot. Our technique allows for easy purification of the quantum dot labeled T7RNA polymerases from the reactants. Also, the conjugation does not alter the functionality of the polymerase; it retains the ability to bind and transcribe.

Enhanced in vitro translation at reduced temperatures using a cold-shock RNA motif

Cell-free synthesis of recombinant proteins has emerged as an alternative method of protein production although protein yields still cannot compete with in vivo expression techniques. In systems based on S30 extracts of Escherichia coli unfavorable side-reactions are involved in limiting protein yields. Therefore, carrying out cell-free reactions at lower temperatures might be beneficial as side reactions should be decreased. In this study we show that by using the 5'-untranslated region of the cold-shock gene cspA from E. coli as mRNA leader in cell-free reactions, the expression temperature can be decreased and simultaneously leads to an increase in protein yields. A compensation for the lower activity of T7 RNA polymerase at lower temperatures enhances protein synthesis even further. Additionally, this 5'-untranslated region also standardizes the optimal expression temperature of different proteins.

Structural studies of arginine induced enhancement in the activity of T7 RNA polymerase

Addition of arginine enhances the activity of the enzyme T7 RNA polymerase. Different methods have been employed to understand the enhancement in the light of arginine induced alteration of the tertiary structure. The increase in activity of the enzyme reaches a maximum value around a concentration of 125mM arginine. Fluorescence, circular dichroism and dynamic light scattering studies indicate an alteration in the tertiary structure of the enzyme. Enthalpy change as a function of input concentration of arginine to a fixed concentration of the enzyme (5μM) shows a dip at 100mM concentration of arginine. Differential scanning calorimetric studies of the denaturation of the enzyme in absence and presence of arginine indicates arginine induced destabilization of the C-terminal domain of the enzyme. Structural alterations induced by arginine have been compared with those induced by the denaturant guanidine hydrochloride.