T7 Pol Research Articles

Protein expression systems combining a flavonoid-inducible promoter and T7 RNA polymerase in Bacillus subtilis.

Recombinant protein production must be tightly controlled when overproduction adversely affects the host bacteria. We developed a flavonoid-inducible T7 expression system in Bacillus subtilis using the qdoI promoter to control the T7 RNA polymerase gene (T7 pol). Using the egfp reporter gene controlled by the T7 promoter in a multicopy plasmid, we confirmed that this expression system is tightly regulated by flavonoids, such as quercetin and fisetin. Altering the qdoI promoter for T7 pol control to its hybrid derivative increased the expression level by 6.6-fold at maximum values upon induction. However, faint expression leakage was observed under a noninducing condition. Therefore, the two expression systems with the original qdoI promoter and the hybrid construct can be used selectively, depending on the high control accuracy or production yield required.

Ultrasensitive electrochemical biosensing strategy for microRNA-21 detection based on homogeneous target-initiated transcription amplification

In this work, we have developed a simple and rapid biosensing method for highly sensitive detection of microRNA-21 (miRNA-21) by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process. MiRNA-21, as a target, recognizes and binds with the corresponding site of the multifunction straight probe, which would trigger extension reaction to form T7 promoters in the presence of Klenow fragment (KF exo−) and dNTPs. The remaining sites of multifunction straight probe serve as templates for transcription amplification based on T7 RNA polymerase (T7 pol.). As a result, a great amount of single-stranded RNA products are obtained, which hybridize with the biotinylated detection probes in the solution, forming transcription products. Finally, the resulted transcription products hybridize with the capture probes immobilized on the surface of electrode, producing electrochemical signal read-out. The developed method achieved a detection limit as low as 0.6fM with a dynamic response range from 1fM to 10nM and could discriminate single-nucleotide differences in microRNA (miRNA) sequences. In addition, the established electrochemical strategy was applied to directly detect miRNA-21 in total RNA extracted from HepG2 cells. Thus, this simple and sensitive electrochemical biosensing method might be a potential alternative tool for the quantitative analysis of miRNAs in physiological fluids, biomedical research and point-of-care testing (POCT) in the future.

T7 RNA polymerase-dependent and -independent systems for cDNA-based rescue of Rift Valley fever virus

Rift Valley fever virus (RVFV) is responsible for large and recurrent outbreaks of acute febrile illness among humans and domesticated animals in Africa. It belongs to the family Bunyaviridae, genus Phlebovirus, and its negative-stranded RNA genome consists of three segments. Here, we report the establishment and characterization of two different systems to rescue the RVFV wild-type strain ZH548. The first system is based on the BHK-21 cell clone BSR-T7/5, which stably expresses T7 RNA polymerase (T7 pol). Rescue of wild-type RVFV was achieved with three T7 pol-driven cDNA plasmids representing the viral RNA segments in the antigenomic sense. The second system involves 293T cells transfected with three RNA pol I-driven plasmids for the viral segments and two RNA pol II-driven support plasmids to express the viral polymerase components L and N. It is known that the 5' triphosphate group of T7 pol transcripts strongly activates the antiviral interferon system via the intracellular RNA receptor RIG-I. Nonetheless, both the T7 pol and the pol I/II system were of similar efficiency. This was even true for the rescue of a RVFV mutant lacking the interferon antagonist nonstructural proteins. Further experiments demonstrated that the unresponsiveness of BHK-21 and BSR-T7/5 cells to T7 pol transcripts is most probably due to a deficiency in the RIG-I pathway. Our reverse genetics systems now enable us to manipulate the genome of RVFV and study its virulence mechanisms. Moreover, the finding that BHK-derived cell lines have a compromised RIG-I pathway may explain their suitability for propagating and rescuing a wide variety of viruses.

Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase

We report fluorescence assays for a functionally important conformational change in bacteriophage T7 DNA polymerase (T7 pol) that use the environmental sensitivity of a Cy3 dye attached to a DNA substrate. An increase in fluorescence intensity of Cy3 is observed at the single-molecule level, reflecting a conformational change within the T7 pol ternary complex upon binding of a dNTP substrate. This fluorescence change is believed to reflect the closing of the T7 pol fingers domain, which is crucial for polymerase function. The rate of the conformational change induced by a complementary dNTP substrate was determined by both conventional stopped-flow and high-time-resolution continuous-flow fluorescence measurements at the ensemble-averaged level. The rate of this conformational change is much faster than that of DNA synthesis but is significantly reduced for noncomplementary dNTPs, as revealed by single-molecule measurements. The high level of selectivity of incoming dNTPs pertinent to this conformational change is a major contributor to replicative fidelity.

Functional Evidence for a Small and Rigid Active Site in a High Fidelity DNA Polymerase: PROBING T7 DNA POLYMERASE WITH VARIABLY SIZED BASE PAIRS

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.

A Unique Region in Bacteriophage T7 DNA Polymerase Important for Exonucleolytic Hydrolysis of DNA

A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E. coli DNA polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 polymerase (pol) Delta144-157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol Delta144-157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol Delta144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3'-5' exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase. Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.

Computer simulation of the chemical catalysis of DNA polymerases: discriminating between alternative nucleotide insertion mechanisms for T7 DNA polymerase.

Understanding the chemical step in the catalytic reaction of DNA polymerases is essential for elucidating the molecular basis of the fidelity of DNA replication. The present work evaluates the free energy surface for the nucleotide transfer reaction of T7 polymerase by free energy perturbation/empirical valence bond (FEP/EVB) calculations. A key aspect of the enzyme simulation is a comparison of enzymatic free energy profiles with the corresponding reference reactions in water using the same computational methodology, thereby enabling a quantitative estimate for the free energy of the nucleotide insertion reaction. The reaction is driven by the FEP/EVB methodology between valence bond structures representing the reactant, pentacovalent intermediate, and the product states. This pathway corresponds to three microscopic chemical steps, deprotonation of the attacking group, a nucleophilic attack on the P(alpha) atom of the dNTP substrate, and departure of the leaving group. Three different mechanisms for the first microscopic step, the generation of the RO(-) nucleophile from the 3'-OH hydroxyl of the primer, are examined: (i) proton transfer to the bulk solvent, (ii) proton transfer to one of the ionic oxygens of the P(alpha) phosphate group, and (iii) proton transfer to the ionized Asp654 residue. The most favorable reaction mechanism in T7 pol is predicted to involve the proton transfer to Asp654. This finding sheds light on the long standing issue of the actual role of conserved aspartates. The structural preorganization that helps to catalyze the reaction is also considered and analyzed. The overall calculated mechanism consists of three subsequent steps with a similar activation free energy of about 12 kcal/mol. The similarity of the activation barriers of the three microscopic chemical steps indicates that the T7 polymerase may select against the incorrect dNTP substrate by raising any of these barriers. The relative height of these barriers comparing right and wrong dNTP substrates should therefore be a primary focus of future computational studies of the fidelity of DNA polymerases.

Replication Slippage of Different DNA Polymerases Is Inversely Related to Their Strand Displacement Efficiency

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.

A T7 RNA polymerase-based system for the construction of Pseudomonas strains with phenotypes dependent on TOL- meta pathway effectors

A general method to construct recombinant Pseudomonas putida (and related bacteria), which transcribe specific genes inserted into their chromosome in response to the presence of alkyl- and halobenzoates, has been developed. The system is based on the ability of the T7 RNA polymerase (T7pol) to initiate transcription from cognate promoter sequences located upstream from cloned genes. A specialized transposon, mini-Tn5 xylS/Pm::T7 pol, was constructed which contains the structural T7 gene 1 downstream from the XylS protein/benzoate-regulated Pm promoter of the meta-operon of the TOL catabolic plasmid. This transposon was stably inserted into the chromosome of a P. putida target strain so that gene 1 is transcribed upon exposure of the bacteria to benzoate effectors of the XylS regulator. Genes whose expression is to be mediated by T7pol are cloned in mini-Tn5 transposons containing T7 promoter sequences upstream from the cloning site and then the hybrid transposons are inserted into different positions in the same chromosome. In this way, expression of the genes cloned within the mini-Tn5 vectors is dependent on the T7pol/XylS/ Pm system. This expression device is particularly well suited for applications in which the expression of two or more genes is to take place in response to a single chemical signal i.e., exposure to certain aromatic compounds.