Papers by Sergei Borukhov
SSRN Electronic Journal
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Biophysical Journal, 2022
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Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In additi... more Type VI CRISPR-Cas systems are the only CRISPR variety that cleaves exclusively RNA1,2. In addition to the CRISPR RNA (crRNA)-guided, sequence-specific binding and cleavage of target RNAs, such as phage transcripts, the type VI effector, Cas13, causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from phage spread3,4. We show here that the principal form of collateral RNA degradation elicited by Cas13a protein from Leptotrichia shahii upon target RNA recognition is the cleavage of anticodons of multiple tRNA species, primarily those with anticodons containing uridines. This tRNA cleavage is necessary and sufficient for bacterial dormancy induction by Cas13a. In addition, Cas13a activates the RNases of bacterial toxin-antitoxin modules, thus indirectly causing mRNA and rRNA cleavage, which could provide a back-up defense mechanism. The identified mode of action of Cas13a resembles that of bacterial anticodon nucleases involved in ...
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Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax... more Bacterial topoisomerase I (TopoI) removes excessive negative supercoiling and is thought to relax DNA molecules during transcription, replication and other processes. Using ChIP-Seq, we show that TopoI of Escherichia coli (EcTopoI) is co-localized, genome-wide, with RNA polymerase (RNAP) in transcription units. Treatment with transcription elongation inhibitor rifampicin leads to EcTopoI relocation to promoter regions, where RNAP also accumulates. When a 14 kDa RNAP-binding EcTopoI C-terminal domain (CTD) is overexpressed, co-localization of EcTopoI and RNAP along the transcription units is reduced. Pull-down experiments directly show that the two enzymes interact in vivo. Using ChIP-Seq and Topo-Seq, we demonstrate that EcTopoI is enriched and in and upstream (within up to 12-15 Kbs) of highly-active transcription units, indicating that EcTopoI relaxes negative supercoiling generated by transcription. Uncoupling of the RNAP-EcTopoI interaction by either overexpression of EcTopoI CT...
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Journal of Biological Chemistry, 1992
The 1342 amino acid long beta subunit of Escherichia coli RNA polymerase includes a dispensable r... 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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CrAss-like phages are a recently described family-level group of viruses that includes the most a... more CrAss-like phages are a recently described family-level group of viruses that includes the most abundant virus in the human gut1,2. Genomes of all crAss-like phages encode a large virion-packaged protein2,3 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)4. Using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a novel DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited, likely pre-virion-packaged state. This conformation is attained with the help of a Cleft-blocking domain that interacts with the active site motif and occupies the RNA-DNA hybrid binding grove. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs involved in RNA interference5,6, although most of phi14:2 RNAP structure (nearly 1,600 residues) map...
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Encyclopedia of Biological Chemistry, 2013
The multisubunit DNA-dependent RNA polymerase (RNAP) mediates transcription – the first step in g... more The multisubunit DNA-dependent RNA polymerase (RNAP) mediates transcription – the first step in gene expression in all cellular organisms. Its basic function is to faithfully synthesize an RNA chain complementary to the transcribed (template) DNA strand in the presence of substrates, ribonucleoside 5′-triphosphates. RNAP structure and function are highly conserved in all three kingdoms of life: bacteria, archaea, and eukaryotes. In bacteria and archaea, a single RNAP enzyme is responsible for the synthesis of all RNAs in the cell, including messenger RNA, ribosomal RNA, transfer RNA, and small noncoding RNA. In eukaryotes, these functions are divided between three related nuclear RNAPs. To orchestrate transcription of ~4000 bacterial genes during cell growth and in response to environmental signals, the enzymatic activity of RNAP is tightly regulated by both intrinsic signals (in DNA template and nascent RNA) and various extrinsic factors (proteins, short peptides, noncoding RNAs, and some small molecules). Extensive genetic, biochemical, and structural studies of RNAP and its regulatory factors conducted in the past 15 years have greatly expanded our knowledge of the organization and function of all major elements of transcription machinery. This article provides a summary of our current understanding of the structure and the mechanisms of action of bacterial RNAPs.
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Bacillus subtilis bacteriophage AR9 employs two strategies for efficient host takeover control an... more Bacillus subtilis bacteriophage AR9 employs two strategies for efficient host takeover control and host antiviral defense evasion – it encodes two unique DNA-dependent RNA polymerases (RNAPs) that function at different stages of virus morphogenesis in the cell, and its double stranded (ds) DNA genome contains uracils instead of thymines throughout1,2. Unlike any known RNAP, the AR9 non-virion RNAP (nvRNAP), which transcribes late phage genes, recognizes promoters in the template strand of dsDNA and efficiently differentiates obligatory uracils from thymines in its promoters3. Here, using structural data obtained by cryo-electron microscopy and X-ray crystallography on the AR9 nvRNAP core, holoenzyme, and a promoter complex, and a variety of in vitro transcription assays, we elucidate a unique mode of uracil-specific, template strand-dependent promoter recognition. It is achieved by a tripartite interaction between the promoter specificity subunit, the core enzyme, and DNA adopting a...
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Nucleic Acids Research, 2018
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Biophysical Journal, 2016
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Nucleic Acids Research
Type II toxin–antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise ... more Type II toxin–antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin–antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin–antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxin...
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ABSTRACTThe Escherichia coli microcin C (McC) and related compounds are potent Trojan-horse pepti... more ABSTRACTThe Escherichia coli microcin C (McC) and related compounds are potent Trojan-horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by non-specific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This non-hydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside of the producing cells, special mechanisms evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by stand-alone genes confer resistance to McC–like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive.IMPORTANCEUncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communicatio...
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Frontiers in Molecular Biosciences
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Nucleic Acids Research, 2017
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Transcription, 2017
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Protein Science, 2017
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Proceedings of the National Academy of Sciences, 2016
Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approa... more Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP–GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused–backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalan...
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Biochem Biophys Res Commun, 1990
The environment of Trp residues of the recombinant human interferons has been studied by the anal... more The environment of Trp residues of the recombinant human interferons has been studied by the analysis of the emission spectra of native and denatured proteins at pH 1.5-8.5 and temperature 10-65 degrees C in the presence and absence of the anionic, cationic and neutral to charge contact quenchers--KI, CsCl and acrylamide, respectively. The obtained data allow to suppose that in IFN-alpha A and IFN-beta 1 Trp141 interacts with Arg145 and one or several from the following residues--Phe124, Ile127, Tyr130, Leu131, whereas Trp77--with Arg33 and Phe36, Phe78, Leu81 or Leu82 (Ile81 or Val82 for IFN-beta 1).
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Biochem Biophys Res Commun, 1990
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Papers by Sergei Borukhov