Dmitry A. Kostyuk

MUTANTS OF T7 RNA-POLYMERASE THAT ARE ABLE TO SYNTHESIZE BOTH RNA AND DNA

A mutant T7 RNA polymerase (T7 RNAP) having two amino‐acid substitutions (Y639F and S641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution S641A loses RNA polymerase activity but is able to synthesize DNA.

Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase

Synthesis of deoxynucleotide‐containing RNA‐like single‐stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter‐containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8–10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1–+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non‐coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG‐containing) was achieved with high yields.

Random mntagenesis of the gene for bacteriophage T7 RNA polymerase

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.

Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction

The mutant T7 RNA polymerase (T7 RNAP), containing two substitutions (Y639F, S641A) was earlier shown to utilize both rNTP and dNTP in a transcription‐like reaction. In this report the ability of the enzyme to catalyze DNA primer extension reaction was demonstrated. The efficiency of the reaction essentially depended on the type of the primer sequence, and was significantly higher if the primer coincided with the T7 promoter non‐coding sequence. In this case the primer extension reaction proceeded along with de novo RNA synthesis. The length of the product did not exceed 8 nucleotides, indicating that the primer extension reaction proceeds according to the mechanism of the T7 RNAP‐catalyzed abortive transcription.

Targeted mutagenesis identifies Asp-569 as a catalytically critical residue in T7 RNA polymerase

In order to look more closely at a well-conserved region in T7 RNA polymerase (T7 RNAP) containing, as shown earlier, the functionally essential residues Pro-563 and Tyr-571, we used targeted mutagenesis to change those residues within this region that are invariant in all single-subunit RNA polymerases, and characterized the mutant enzymes in vitro. The most interesting finding of this study was the crucial importance of the acidic group of Asp-569. In addition, we have shown that the phenolic ring is the most significant functional group of Tyr-571, with the hydroxy group also contributing to promoter binding.

On the functional role of the Tyr-639 residue of bacteriophage T7 RNA polymerase

Substitution or Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity. This mutation does not affect the enzyme‐promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain.

Tyr-571 is involved in the T7 RNA polymerase binding to its promoter

The in vitro studies of three T7 RNA polymerase point mutants suggest that substitutions of Ala and Thr for Pro‐563 and of Ser for Tyr‐571 have little effect on the enzyme catalytic competence, but result in its inability to utilize the promoter. Both P563A and P563T mutants retain the promoter‐binding ability, whereas the promoter affinity of the Y571S mutant drops drastically.

New Non-nucleoside Inhibitors of Hepatitis C Virus RNA-Dependent RNA Polymerase

Recombinant RNA-dependent RNA polymerase of hepatitis C virus was purified using a bacterial expression system (Escherichia coli). The system for enzyme activity detection was optimized. The maximum activity was achieved when the reaction was carried out at 30°C in the presence of 3 mM Mg2+ or 0.75 mM Mn2+. Among α- and β-pyrogallaldehydes, effective inhibitors were found. It was shown that they acted at the primer elongation stage, and their binding to the protein is reversible.

Synthesis of Mixed Ribo/Deoxyribopolynucleotides by Mutant T7 RNA Polymerase

Synthesis of deoxynucleotide-containing RNA-like single-stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter-containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8-10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1-+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non-coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG-containing) was achieved with high yields.