Affinity labelling with aldehyde‐containing analogs of initiation substrates of nuclear fraction of tick‐borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which... more
Affinity labelling with aldehyde‐containing analogs of initiation substrates of nuclear fraction of tick‐borne encephalitis virus (TBEV) infected cells results in a labelling of a single polypeptide with a molecular mass of 68 kDa which was immunologically identified as TBEV NS3 protein. A single‐hit hydroxylamine hydrolysis, using limited and long‐term CNBr cleavages allowed one to identify Lys1800 and/or Lys1803 as the label attachment sites. These amino acid residues are situated in the proximity of the ‘B’‐site of NTP‐binding motif of viral RNA replicase.
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E. coli RNA polymerase was selectively labelled in the presence of promoters at a histidine residue of the beta-subunit by treatment with GDP beta-imidazolide and then with [alpha-32P]UTP (or [alpha-33P]UTP). Partial cyanogen bromide... more
E. coli RNA polymerase was selectively labelled in the presence of promoters at a histidine residue of the beta-subunit by treatment with GDP beta-imidazolide and then with [alpha-32P]UTP (or [alpha-33P]UTP). Partial cyanogen bromide cleavage of the labelled polypeptide afforded a series of "single-hit" labelled peptides, the electrophoretic pattern of which suggested that the labelling site was His1237. This conclusion was confirmed by a similar pattern obtained with products of the cyanogen bromide cleavage of a radioactive peptide obtained by the limited trypsinolysis (C-terminal peptide consisting of 423 amino acid residues). Interpretation of our earlier results in favour of His1116 as the labelling point (Dokl. Acad. nauk SSSR, 1985, v. 281, p. 723) was incorrect due to the electrophoretic "compression" of three labelled peptide bands.
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RNA polymerase A, B and C from yeast were modified with 4-hydroxybenzaldehyde which had been esterified to the terminal phosphate of ATP and subsequently reduced with NaBH4. Upon incubation with a-[32p]UTP, the second largest subunits... more
RNA polymerase A, B and C from yeast were modified with 4-hydroxybenzaldehyde which had been esterified to the terminal phosphate of ATP and subsequently reduced with NaBH4. Upon incubation with a-[32p]UTP, the second largest subunits A135, B150 or C128 became heavily labelled in a template-dependent reaction which exploits the catalytic activity of the modified enzymes. In the case of subunit B150, the covalently bound label was shown to consist of a trinucleotide and ApU, respectively. The labelling was prevented by a-amanitine at concentrations similar to those used to inhibit RNA synthesis in vitro. These results indicate a very similar function of the second largest subunit in the reaction catalysed by the three nuclear RNA polymerases. If the reduction with NaBH4 was carried out after the enzymatic condensation, both large subunits of enzyme A and B became labelled.
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We report a new observation of the role of Escherichia coli single-strand DNA binding protein (SSB) in synthesis of primer RNA (pRNA) catalyzed by.E.coli primase on the SSB-coated phage G4oric template. Using a set of ATP priming... more
We report a new observation of the role of Escherichia coli single-strand DNA binding protein (SSB) in synthesis of primer RNA (pRNA) catalyzed by.E.coli primase on the SSB-coated phage G4oric template. Using a set of ATP priming substrates with reactive groups attached to the 5' gamma-phosphate on different length "arms", we have demonstrated that, in the primase/SSB/G4oric pRNA synthesis complex, ATP cross-linked to both primase and SSB could be equally utilized as initiating nucleotide for pRNA synthesis. The distance between SSB surface and alpha-phosphorus of the priming substrate was estimated to be less than 7 A. ATP cross-linked to primase and SSB can be further elongated in the presence of other NTPs, giving almost identical patterns of covalently attached pRNAs of up to 12 nucleotides in length. The regions of primase and SSB with cross-linked ATP that can be used for pRNA synthesis are, therefore, arranged in a similar way relative to the active center of pRNA synthesis. The pRNA covalently linked to SSB was localized, mapping between Met48 and Trp88. This observation raises the possibility that SSB may play an active role in the initiation of pRNA synthesis in this system.
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The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction of gene expression. Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in the beta... more
The active center of DNA-dependent RNA polymerase performs the principal biochemical reaction of gene expression. Using cross-linkable substrate analogs and site-directed mutations, two evolutionarily invariant amino acids in the beta subunit of the Escherichia coli enzyme (Lys1065 and His1237) were mapped close to the binding site of the priming substrate of the reaction. Surprisingly, the mutational substitution of these residues (Lys1065----Arg and His1237----Ala) did not inactivate the catalytic function, but inhibited transition from the initiation to the elongation stage of transcription.
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His1237 in the beta subunit of Escherichia coli RNA polymerase marks the "5' face" of the active center since it can be cross-linked to the gamma-phosphate of the priming substrate. It is demonstrated that RNA... more
His1237 in the beta subunit of Escherichia coli RNA polymerase marks the "5' face" of the active center since it can be cross-linked to the gamma-phosphate of the priming substrate. It is demonstrated that RNA chains up to 9 nucleotides in length can be synthesized using His1237-cross-linked nucleotide as a primer. Thus, a substantial mass of RNA can be accommodated in the active center between His1237 and the site of catalysis that remains juxtaposed to the growing 3' end. The apparent "filling" of the active center with RNA precedes promoter clearance and suggests a mechanism of coupling between catalysis and saltatory translocation of RNA polymerase.
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Abstract-Luminescent lanthanide probes possess long emission lifetime, which enables their hypersensitive detection in time-resolved mode that avoids short-lived background fluorescence. Sharply spiked emission spectra of lanthanide... more
Abstract-Luminescent lanthanide probes possess long emission lifetime, which enables their hypersensitive detection in time-resolved mode that avoids short-lived background fluorescence. Sharply spiked emission spectra of lanthanide probes and large Stokes shifts further enhance detection sensitivity, which is about 1000 fold higher than that for regular fluorescent probes. The wide spread of this promising technology is limited by high cost of commercially available compounds, which is mainly due to their complex structure and laborious synthetic procedure. In this study, new efficient strategies to simplify the synthesis of the probes were explored. New lanthanide chelates, containing click-, and amine-reactive cross-linking groups, which are highly bright and can be produced with high yield, are synthesized and characterized.
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The spread of drug-resistant bacteria represents one of the most significant medical problems of our time. Bacterial fitness loss associated with drug resistance can be counteracted by acquisition of secondary mutations, thereby enhancing... more
The spread of drug-resistant bacteria represents one of the most significant medical problems of our time. Bacterial fitness loss associated with drug resistance can be counteracted by acquisition of secondary mutations, thereby enhancing the virulence of such bacteria. Antibiotic rifampicin (Rif) targets cellular RNA polymerase (RNAP). It is potent broad spectrum drug used for treatment of bacterial infections. We have investigated the compensatory mechanism of the secondary mutations alleviating Rif resistance (Rifr) on biochemical, structural and fitness indices. We find that substitutions in RNAP genes compensating for the growth defect caused by βQ513P and βT563P Rifr mutations significantly enhanced bacterial relative growth rate. By assaying RNAP purified from these strains, we show that compensatory mutations directly stimulated basal transcriptional machinery (2–9-fold) significantly improving promoter clearance step of the transcription pathway as well as elongation rate. Molecular modeling suggests that compensatory mutations affect transcript retention, substrate loading, and nucleotidyl transfer catalysis. Strikingly, one of the identified compensatory substitutions represents mutation conferring rifampicin resistance on its own. This finding reveals an evolutionary process that creates more virulent species by simultaneously improving the fitness and augmenting bacterial drug resistance. Open in a separate windowGraphical AbstractGrowth rates at 20 °C and 37 °C for the E. coli strains studied in this work (top) and the activity of RNA polymerase purified from these strains in various in vitro assays at the same temperatures (bottom).
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The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov,... more
The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable region (residues 940-1040) that is absent in homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria (Borukhov, S., Severinov, K., Kashlev, M., Lebedev, A., Bass, I., Rowland, G. C., Lim, P.-P., Glass, R. E., Nikiforov, V., and Goldfarb, A. (1991) J. Biol. Chem. 266, 23921-23926). Genetic disruption of this region by in-frame deletion or insertion sensitizes the beta subunit in assembled RNA polymerase molecules to attack by trypsin. We demonstrate that RNA polymerase with the beta polypeptide cleaved in the dispensable region retains normal in vitro activity. Moreover, the RNA polymerase activity is completely restored after denaturation and reconstitution of the enzyme carrying cleaved beta subunit indicating that its carboxyl- and amino-terminal parts fold and assemble into RNA polymerase as separate entities.
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Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such... more
Incubation of the Klenow fragment of E. coli DNA polymerase I with [alpha-32P] dNTP (or NTP) results in the covalent radiolabelling of the enzyme, the bond being stable in acid (pH 2) and alkaline (pH 12) conditions and nucleophiles, such as beta-mercaptoethylamine, efficiently inhibiting the labelling. It is suggested that radiolabelling of the enzyme is the result of formation of chemically active products of the radiolysis of [alpha-32P]NTP (which are likely to be radicals). Non-radioactive NTP hinder the labelling, whereas Mg2+ and polynucleotide do not affect it. Cleavage of the enzyme by hydroxylamine and cyanogen bromide and analysis of gel-electrophoretic patterns of the cleavage products led to conclusion that 32P-label is located between Gly-544 and Met-647.
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Highly selective affinity labeling of a DNA-polymerase alpha-primase complex from human placenta by o-formylphenyl esters of ATP, ADP and AMP was performed in a two-step procedure in which a substrate analog attached to the active center... more
Highly selective affinity labeling of a DNA-polymerase alpha-primase complex from human placenta by o-formylphenyl esters of ATP, ADP and AMP was performed in a two-step procedure in which a substrate analog attached to the active center was elongated by radioactive ATP. If the covalent attachment is performed in the presence of poly(dT) template, the ATP esters modify selectively the delta subunit of the complex. If poly(dT) is added after the covalent binding of the reagent, both delta and gamma subunits become labeled. With the o-formylphenyl ester of AMP the delta-subunit is modified. The ADP ester modifies both the delta and gamma subunit in the presence and absence of template. It is shown that formylphenyl ester of ATP is not the substrate in the reaction of elongation catalyzed by primase. The data obtained suggest the binding site of initiating substrate to be located in the region of contact of the two subunits of primase. The role of the template in the formation of the active site is discussed.
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Antimicrobial-resistant (AMR) infections pose a major threat to global public health. Similar to other AMR pathogens, both historical and ongoing drug-resistant tuberculosis (TB) epidemics are characterized by transmission of a limited... more
Antimicrobial-resistant (AMR) infections pose a major threat to global public health. Similar to other AMR pathogens, both historical and ongoing drug-resistant tuberculosis (TB) epidemics are characterized by transmission of a limited number of predominant Mycobacterium tuberculosis ( Mtb ) strains. Understanding how these predominant strains achieve sustained transmission, particularly during the critical period before they are detected via clinical or public health surveillance, can inform strategies for prevention and containment. In this study, we employ whole-genome sequence (WGS) data from TB clinical isolates collected in KwaZulu-Natal, South Africa to examine the pre-detection history of a successful strain of extensively drug-resistant (XDR) TB known as LAM4/KZN, first identified in a widely reported cluster of cases in 2005. We identify marked expansion of this strain concurrent with the onset of the generalized HIV epidemic 12 y prior to 2005, localize its geographic ori...
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The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting... more
The path of the nucleic acids through a transcription elongation complex was tracked by mapping cross-links between bacterial RNA polymerase (RNAP) and transcript RNA or template DNA onto the x-ray crystal structure. In the resulting model, the downstream duplex DNA is nestled in a trough formed by the β′ subunit and enclosed on top by the β subunit. In the RNAP channel, the RNA/DNA hybrid extends from the enzyme active site, along a region of the β subunit harboring rifampicin resistance mutations, to the β′ subunit “rudder.” The single-stranded RNA is then extruded through another channel formed by the β-subunit flap domain. The model provides insight into the functional properties of the transcription complex.
Research Interests: Science, Biology, RNA polymerase, Medicine, Gene expression, and 15 moreMultidisciplinary, DNA, Mutation, Nucleic acid hybridization, Bacteria, DNA hybridization, Mechanism, Elongation, Enzyme activity, Protein Conformation, Crystalline Structure, Nucleic Acid Conformation, Nucleic Acid, Functional Properties, and binding sites
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Research Interests: Biology, Biological Chemistry, Medicine, Macromolecular X-Ray Crystallography, Biological Sciences, and 13 moreMutation, Escherichia coli, CHEMICAL SCIENCES, Anti-Bacterial Agents, Dna Gyrase, Microbial Sensitivity Tests, Ciprofloxacin, Protein Binding, Nucleic Acid Conformation, Fluoroquinolones, Molecular Structure, Mycobacterium smegmatis, and Medical and Health Sciences
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Research Interests: Catalysis, RNA polymerase, Biological Sciences, Environmental Sciences, Magnesium, and 11 moreEscherichia coli, Nucleic Acids, D-Aspartic Acid, Amino Acid Profile, Amino Acid Sequence, Base Sequence, Amino Acid Substitution Rates, Catalytic Activity, Structural model, Nucleic Acid, and Molecular Sequence Data
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Recognition of the -10 promoter consensus element by region 2 of the bacterial RNA polymerase sigma subunit is a key step in transcription initiation. sigma also functions as an elongation factor, inducing transcription pausing by... more
Recognition of the -10 promoter consensus element by region 2 of the bacterial RNA polymerase sigma subunit is a key step in transcription initiation. sigma also functions as an elongation factor, inducing transcription pausing by interacting with transcribed DNA non-template strand sequences that are similar to the -10 element sequence. Here, we show that the region 1.2 of Escherichia coli sigma70, whose function was heretofore unknown, is strictly required for efficient recognition of the non-template strand of -10-like pause-inducing DNA sequence by sigma region 2, and for sigma-dependent promoter-proximal pausing. Recognition of the fork-junction promoter DNA by RNA polymerase holoenzyme also requires sigma region 1.2 and thus resembles the pause-inducing sequence recognition. Our results, together with available structural data, support a model where sigma region 1.2 acts as a core RNA polymerase-dependent allosteric switch that modulates non-template DNA strand recognition by ...
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Inorganic Pi is involved in all major biochemical pathways. Here we describe a previously unreported activity of Pi. We show that Pi and its structural mimics, vanadate and arsenate, enhance nascent transcript cleavage by RNA polymerase... more
Inorganic Pi is involved in all major biochemical pathways. Here we describe a previously unreported activity of Pi. We show that Pi and its structural mimics, vanadate and arsenate, enhance nascent transcript cleavage by RNA polymerase (RNAP). They engage an Mg2+ ion in catalysis and activate an attacking water molecule. Pi, vanadate, and arsenate stimulate the intrinsic exonuclease activity of the enzyme nearly 2,000-fold at saturating concentrations of the reactant anions and Mg2+. This enhancement is comparable to that of specialized transcript cleavage protein factors Gre and TFIIS (3,000- to 4,000-fold). Unlike these protein factors, Pi and its analogs do not stimulate endonuclease transcript cleavage. Conversely, the protein factors only marginally enhance exonucleolytic cleavage. Pi thus complements cellular protein factors in assisting hydrolytic RNA cleavage by extending the repertoire of RNAP transcript degradation modes.
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Fungal keratitis is a leading cause of ocular morbidity and blindness in developing countries. Diagnosing fungal keratitis currently relies on a comparative evaluation of corneal biopsy or scraping using a direct microscopy and culture... more
Fungal keratitis is a leading cause of ocular morbidity and blindness in developing countries. Diagnosing fungal keratitis currently relies on a comparative evaluation of corneal biopsy or scraping using a direct microscopy and culture results. These methods not only carry the risk of developing complications due to the invasive tissue sampling but also are largely limited by diagnostic speed and accuracy, making it difficult to initiate timely appropriate antifungal therapy. Therefore, rapid and noninvasive diagnostic tools are a pressing need for improved outcomes for fungal keratitis. Taking advantage of the highly specific fungal cell targeting properties of caspofungin, we have developed a fluorescent chemical probe with high selectivity against fungal pathogens. Utilizing fluorescence imaging technology, we have demonstrated a highly specific and sensitive detection of Aspergillus in a fungal keratitis model in mice as early as 5 min post-topical application of the probe. Our ...
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The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that... more
The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5′ and the 3′ ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3′ end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5′ end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the...
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This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Modified synthetic evolution of ligands through exponential enrichment (SELEX) was used to discover a DNA... more
This technical report describes the development of an aptamer for sensing azole antifungal drugs during therapeutic drug monitoring. Modified synthetic evolution of ligands through exponential enrichment (SELEX) was used to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding to its target molecule, as shown through fluorescence anisotropy-based binding measurements. Experiments using circular dichroism spectroscopy revealed a unique G-quadruplex structure that was essential and specific for binding to the azole antifungal target. Aptamer-functionalized graphene field effect transistor (GFET) devices were created and used to measure the strength of binding of azole antifungals to this surface. In total, this aptamer and the supporting sensing platform provide a valuable tool for therapeutic drug monitoring of patients with invasive fungal infections. IMPORTANCE We have developed the first aptamer directed ...