Development of the system ensuring a high-level expression of hepatitis C virus nonstructural NS5B and NS5A proteins.

The plasmid pET-21d-2c-5BDelta55 effectively expressing a C-terminally truncated form (NS5BDelta55) of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) was constructed. It was derived from pET-21d-5BDelta55 plasmid and contained six mutations in the ATG-start codon region and an additional cistron upstream the target gene. The C-terminally His-tagged NS5BDelta55 protein was expressed in Rosetta(DE3) Escherichia coli strain bearing an additional pRARE plasmid encoding extra copies of rare tRNAs.

Reverse transcriptase-based DNA vaccines against drug-resistant HIV-1 tested in a mouse model.

Drug resistance is becoming a problem in the treatment of the human immunodeficiency virus type one (HIV-1). To obtain therapeutic DNA vaccines that would target multiple drug-resistance (DR) mutations, we cloned genes for DR HIV-1 reverse transcriptase (RT) and codon-optimized synthetic genes encoding clusters of human CTL epitopes located at the sites of DR-mutations (RT minigenes) and antibody and CTL-epitope tags. Expression of RT genes/minigenes in eukaryotic cells was confirmed by Western blotting and immunofluoresence staining with RT- or tag-specific antibodies.

Functional expression of eukaryotic polypeptide chain release factors 1 and 3 by means of baculovirus/insect cells and complex formation between the factors.

Translation termination in eukaryotes is governed by termination codons in mRNA and two release factors, eRF1 and eRF3. In this work, human eRF1 and eRF3 have been produced in insect cells using a recombinant baculovirus expression system for the corresponding human cDNAs. Purification of eRF1 has led to a homogeneous 50-kDa protein active in promoting ribosome-dependent and termination-codon-dependent hydrolysis of formylmethionyl-tRNAf(Met).

The DNA polymerase-encoding gene from a thermoacidophilic archaeon Sulfolobus acidocaldarius.

We have cloned, sequenced and characterized the gene encoding a DNA polymerase from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sac). The putative transcription promoter and terminator elements, as well as a potential ribosome-binding site (rbs), have been identified in the flanking regions. One large open reading frame (ORF) found in the sequenced portion of the Sac genome encodes a protein of 875 amino acids (aa).

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.

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.

Interactions of the HIV-1 reverse transcriptase 'AZT-resistant' mutant with substrates and AZT-TP.

To investigate the biochemical basis of the HIV-1 resistance to AZT we obtained the RT mutant containing four amino acid substitutions by an oligonucleotide-directed mutagenesis technique. Enzymatic properties of the wild type and mutant RTs were compared. 'AZT-resistant' mutations in RT were shown to be associated with the reduced capability of AZT-TP to block the DNA- but not RNA-directed DNA synthesis.

Random mutagenesis 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.

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.

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

Substitution of 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.

Lys631 residue in the active site of the bacteriophage T7 RNA polymerase. Affinity labeling and site-directed mutagenesis.

A highly selective affinity labeling of T7 RNA polymerase with the o-formylphenyl ester of GMP and [alpha-32P]UTP was carried out. The site of the labeling was located using limited cleavages with hydroxylamine, bromine, N-chlorosuccinimide and cyanogene bromide and was identified as the Lys631 residue. Site-directed mutagenesis using synthetic oligonucleotides was used to substitute Lys631 by a Gly, Leu or Arg residue.

Inactivation of bacteriophage T7 DNA-dependent RNA polymerase by 5'-p-fluorosulfonylbenzoyladenosine. Identification of the modification site and the effect of the modification on enzyme action.

Bacteriophage T7 RNA polymerase was covalently modified by 5'-[4-fluorosulfonyl)benzoyl]adenosine (4-FSO2BzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the phi 10 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4-FSO2BzAdo within the covalent enzyme-inhibitor complex restores the RNA synthesis at a lower rate.

The mechanism of pyrophosphorolysis of RNA by RNA polymerase. Endowment of RNA polymerase with artificial exonuclease activity.

DNA-directed RNA polymerase from Escherichia coli can break down RNA by catalysing the reverse of the reaction: NTP + (RNA)n = (RNA)n+1 + PPi where n indicates the number of nucleotide residues in the RNA molecule, to yield nucleoside triphosphates. This reaction requires the ternary complex of the polymerase with template DNA and the RNA that it has synthesized. It is now shown that methylenebis(arsonic acid) [CH2(AsO3H2)2], arsonomethylphosphonic acid (H2O3As-CH2-PO3H2) and arsonoacetic acid (H2O3As-CH2-CO2H) can replace pyrophosphate in this reaction.