Olga N. Koroleva

Is transketolase-like protein, TKTL1, transketolase?

Until recently it was assumed that the transketolase-like protein (TKTL1) detected in the tumor tissue, is catalytically active mutant form of human transketolase (hTKT). Human TKT shares 61% sequence identity with TKTL1. And the two proteins are 77% homologous at the amino acid level. The major difference is the absence of 38 amino acid residues in the N-terminal region of TKTL1. Site-specific mutagenesis was used for modifying hTKT gene; the resulting construct had a 114-bp deletion corresponding to a deletion of 38 amino acid residues in hTKT protein. Wild type hTKT and mutant variant (DhTKT) were expressed in Escherichia coli and isolated using Ni-agarose affinity chromatography. We have demonstrated here that DhTKT is devoid of transketolase activity and lacks bound thiamine diphosphate (ThDP). In view of these results, it is unlikely that TKTL1 may be a ThDP-dependent protein capable of catalyzing the transketolase reaction, as hypothesized previously.

A system for heterologous expression and isolation of Escherichia coli RNA polymerase and its components

A set of plasmid vectors for expression of all major Escherichia coli RNA polymerase subunits as fusion proteins with intein-and chitin-binding domains, allowing protein purification in accordance with IMPACT technology, was constructed. It is demonstrated that the fusion subunits α, β, or β′ in conjunction with the natural subunits α, β, β′, and σ can participate in RNA polymerase assembly in vivo, providing affinity-based isolation of the enzyme. Functional activity of the enzyme preparations was demonstrated in the experiments on in vitro transcription and promoter complex formation. With the use of IMPACT technology, σ70 subunit can be isolated as an individual protein without admixture of RNA polymerase.

Bisbenzimidazole derivatives as potent inhibitors of the trypsin-like sites of the immunoproteasome core particle

In this study, a monomeric (MB) and a dimeric (DB) bisbenzimidazoles were identified as novel proteasome inhibitors of the trypsin-like activity located on β2c sites of the constitutive 20S proteasome (IC50 values at 2-4 μM range). Remarkably, they were further shown to be 100- and 200-fold more potent inhibitors of the immunoproteasome trypsin-like activity (β2i sites, IC50=24 nM) than of the homologous constitutive activity. Molecular models of inhibitor/enzyme complexes in the two types of trypsin-like sites and corresponding computed binding energy values corroborated kinetic data. Different binding modes were suggested for MB and DB to the β2c and β2i trypsic sites. Each pointed to better contacts of the ligand inside the β2i active site than for β2c site. MB and DB represent the first selective inhibitors of the immunoproteasome trypsin-like activity described to date and can be considered as prototypes for inhibiting this activity.

Trifluoromethyldiazirine-Containing dUTP: Synthesis and Application in DNA/Protein Crosslinking

Abstract The 5-[N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-3-aminoallyl]-2′-deoxyuridine-5′-triphosphate was synthesized via acylation of 5-aminoallyl-2′-deoxyuridine-5′-triphosphate with 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate N-hydroxysuccinimide. It was used for the preparation of 30 bp ATFMD-DNA coding for promoter sequence. UV-Irradiation (365 nm) of the specific complex of this duplex and E. coli RNA polymerase leads to the effective crosslinking DNA with all protein subunits.

Effect of DNA bending on transcriptional interference in the systems of closely spaced convergent promoters

BACKGROUND Over the past years there are increasing evidences that the interplay between two molecules of RNA polymerases, initiating transcription from promoters, oriented in opposite (convergent) directions, can serve as a regulatory factor of gene expression. The data concerning the molecular mechanisms of this so-called transcriptional interference (TI) are not well understood. METHODS The interaction of RNA polymerase with circular DNA templates, containing the convergent promoters, was investigated in a series of in vitro transcription assays and atomic force microscopy (AFM). RESULTS In this work, to study the mechanisms of transcription interference a series of plasmids with oppositely oriented closely spaced artificial promoters, recognized by Escherichia coli RNA polymerase, was constructed. The constructs differ in promoter structure and distance between the transcription start sites. We have demonstrated that the transcripts ratio (RNA-R/RNA-L) and morphology of convergent open promoter complexes (OPC) are highly dependent on the interpromoter distance. CONCLUSIONS The obtained results allowed us to suggest the novel model of TI, which assumes the DNA bending upon binding of RNA polymerase with promoters and explains the phenomenon of complete inactivation of weaker promoter by the stronger one. GENERAL SIGNIFICANCE The results show that the conformational transitions in DNA helix, associated with DNA bending upon binding of RNA polymerase with promoters, play crucial role in OPC formation in the systems with convergent promoters.

AFM study of Escherichia coli RNA polymerase σ70 subunit aggregation

UNLABELLED The self-assembly of Escherichia coli RNA polymerase σ⁷⁰ subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ⁷⁰ subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ⁷⁰ polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ⁷⁰ subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ⁷⁰ subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. FROM THE CLINICAL EDITOR In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ⁷⁰( subunit was investigated using atomic force microscopy and other complementary approaches.

Effects of substitutions at position 180 in the Escherichia coli RNA polymerase σ70 subunit

In order to investigate the role of His180 residue, located in the non-conserved region of the σ70 subunit of Escherichia coli RNA polymerase, two mutant variants of the protein with substitutions for either alanine or glutamic acid were constructed and purified using the IMPACT system. The ability of mutant σ70 subunits to interact with core RNA polymerase was investigated using native gel-electrophoresis. The properties of the corresponding reconstituted holoenzymes, as provided by gel shift analysis of their complexes with single- and double-stranded promoter-like DNA and by in vitro transcription experiments, allowed one to deduce that His180 influences several steps of transcription initiation, including core binding, promoter DNA recognition and open complex formation.

Purification of core enzyme of Escherichia coli RNA polymerase by affinity chromatography

A method for isolation of a highly purified preparation of E. coli RNA polymerase core enzyme was developed based on IMPACT technology and dissociation of the RNA polymerase complex with σ70 subunit. Washing of the immobilized RNA polymerase with 5–10 mM solution of glutamate (pH 5.0–5.5) completely removed the σ70 subunit from the holoenzyme and decreased amounts of protein admixtures. The possibility of reconstruction of the RNA polymerase holoenzyme directly on the affinity column was demonstrated. Activities of the resulting RNAP core enzyme preparations were tested by in vitro transcription. Some amino acids and their mixtures were shown to influence the in vitro transcription. The findings indicate that changes in the transcription efficiency in the presence of amino acids should be associated with a specific destruction of the interaction between σ70 subunit and the core enzyme.

Heterologous expression and isolation of influenza A virus nuclear export protein NEP

Influenza A virus nuclear export protein NEP (NS2, 14.4 kDa) plays a key role in various steps of the virus life cycle. Highly purified protein preparations are required for structural and functional studies. In this study, we designed a series of Escherichia coli plasmid constructs for highly efficient expression of the NEP gene under control of the constitutive trp promoter. An efficient method for extraction of NEP from inclusion bodies based on dodecyl sulfate treatment was developed. Preparations of purified NEP with either N-or C-terminal (His)6-tag were obtained using Ni-NTA agarose affinity chromatography with yield of more than 20 mg per liter of culture. According to CD data, the secondary structure of the proteins matched that of natural NEP. A high propensity of NEP to aggregate over a wide range of conditions was observed.

Interaction of Escherichia coli RNA Polymerase with Artificial Promoters, Containing Nonnucleotide Spacers

To study the functional role of the spacer region between two consensus −10 and −35 elements of promoters, recognized by E. coli RNA polymerase, the model promoter-like DNA duplexes containing nonnucleotide inserts (mimicking 17-mer spacer) either in one or both strands, were constructed. The modified duplexes can form the heparin-resistant binary complexes with RNA polymerase. The DNA duplex with nonnucleotide insert in the template strand can specifically direct the synthesis of mRNA in the in vitro run-off transcription assays.