Georgiy A Belogurov

University of Turku | UTU · Department of Life Technologies

Formycin A (FOR) and pyrazofurin A (PYR) are nucleoside analogs with antiviral and antitumor properties. They are known to interfere with nucleic acid metabolism, but their direct effect on transcription is less understood. We explored how RNA polymerases (RNAPs) from bacteria, mitochondria, and viruses utilize FOR, PYR, and oxidized purine nucleot...

RNA polymerase (RNAP) mediates the synthesis of an RNA copy of the template DNA—the first and often decisive step in gene expression. All cellular RNAPs possess an additional capacity to cleave nucleotides from the 3’ end of the nascent RNA. This ability potentially enhances the efficiency and accuracy of transcription, enabling RNAP to maintain pr...

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a β-barrel, which binds the ribosome. Here...

RfaH, a paralog of the universally conserved NusG, binds to RNA polymerases (RNAP) and ribosomes to activate expression of virulence genes. In free, autoinhibited RfaH, an α-helical KOW domain sequesters the RNAP-binding site. Upon recruitment to RNAP paused at an ops site, KOW is released and refolds into a β-barrel, which binds the ribosome. Our...

Transcription is the first and most highly regulated step in gene expression. Experimental techniques for monitoring transcription are, thus, important for studying gene expression and gene regulation as well as for translational research and drug development. Fluorescence methods are often superior to other techniques for real-time monitoring of b...

Showdomycin produced by Streptomyces showdoensis ATCC 15227 is a C-nucleoside microbial natural product with antimicrobial and cytotoxic properties. The unique feature of showdomycin in comparison to other nucleosides is its maleimide base moiety, which has the distinct ability to alkylate nucleophilic thiol groups by a Michael addition reaction. I...

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)–σ⁷⁰ holoenzyme to the promoter DNA. This initial RNAP–promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; the...

DNA lesions threaten cell life and must be repaired to maintain genome integrity. During transcription, RNA polymerases actively scan DNA to find lesions and trigger their repair. In growing eukaryotic cells, about 60% of the total transcriptional activity involves the synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I), a 14-subunit macr...

The expression of most bacterial genes commences with the binding of RNA polymerase (RNAP)-σ70 holoenzyme to the promoter DNA. This initial RNAP-promoter closed complex undergoes a series of conformational changes, including the formation of a transcription bubble on the promoter and the loading of template DNA strand into the RNAP active site; the...

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2′dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2′OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2′dNTPs, whereas selectivity mechanism...

Cellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD...

How to stop RNA polymerase Timely and tunable cessation of RNA synthesis is vital for cellular homeostasis. RNA helicases such as the archetypal termination factor r actively dismantle transcription complexes, but the transitory nature of termination makes the process hard to study structurally. Said et al. assembled ρ-bound transcription complexes...

Cellular RNA polymerases can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, or enter dormancy. How RNA polymerase recycling into active states is achieved and balanced with quiescence remains elusive. We structurally analyzed Bacillus subtilis RNA polymerase bound to the NTPase HelD. HelD has two long arm...

RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanism...

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known...

Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the met...

Multi-subunit DNA-dependent RNA polymerases synthesize all classes of cellular RNAs, ranging from short regulatory transcripts to gigantic messenger RNAs. RNA polymerase has to make each RNA product in just one try, even if it takes millions of successive nucleotide addition steps. During each step, RNA polymerase selects a correct substrate, adds...

All cellular RNA polymerases (RNAP) occasionally backtrack along the template DNA as part of transcriptional proofreading and regulation. Here, we studied the mechanism of RNAP backtracking by one nucleotide using two complementary approaches that allowed us to precisely measure the occupancy and lifetime of the backtracked state. Our data show tha...

All cellular RNA polymerases (RNAP) occasionally backtrack along the template DNA as part of transcriptional proofreading and regulation. Here, we studied the mechanism of RNAP backtracking by one nucleotide using two complementary approaches that allowed us to precisely measure the occupancy and lifetime of the backtracked state. Our data show tha...

Significance DNA lesions threaten cellular life and must be repaired to maintain genome integrity. During transcription, RNA polymerases (RNAPs) actively scan DNA to find bulky lesions and trigger their repair. In growing eukaryotic cells, most transcription involves synthesis of ribosomal RNA by RNAP I (Pol I), and Pol I activity thus influences s...

Universally conserved NusG/Spt5 factors reduce RNA polymerase pausing and arrest. In a widely accepted model, these proteins bridge the RNA polymerase clamp and lobe domains across the DNA channel, inhibiting the clamp opening to promote pause‐free RNA synthesis. However, recent structures of paused transcription elongation complexes show that the...

In this issue of Molecular Cell, Guo et al. (2018) and Kang et al. (2018) report structures of paused transcription complexes in which asynchronous translocation inhibits nucleotide addition, allowing for global rearrangements in RNA polymerase stabilized by RNA hairpin and NusA. In this issue of Molecular Cell, Guo et al. (2018) and Kang et al. (2...

Nucleoside antibiotics are a large class of pharmaceutically relevant chemical entities, which exhibit a broad spectrum of biological activities. Most nucleosides belong to the canonical N-nucleoside family, where the heterocyclic unit is connected to the carbohydrate through a carbon-nitrogen bond. However, atypical C-nucleosides have been isolate...

Significance The nontemplate DNA strand in the transcription bubble interacts with RNA polymerase and accessory factors to control initiation, elongation, transcription-coupled repair, and translation. During initiation, σ subunit interactions with the nontemplate DNA regulate promoter complex formation and lifetime, abortive synthesis, and start s...

NusG-TEC model.DOI: http://dx.doi.org/10.7554/eLife.18096.022

Model employed for fitting of nucleotide addition and translocation data. DOI: http://dx.doi.org/10.7554/eLife.18096.027

Model employed for fitting equilibrium titration with TGT. DOI: http://dx.doi.org/10.7554/eLife.18096.029

Model employed for fitting pyrophosphorolysis, backtracking, RNA cleavage and dinucleotide release data. DOI: http://dx.doi.org/10.7554/eLife.18096.028

Model employed for fitting NusG binding kinetics. DOI: http://dx.doi.org/10.7554/eLife.18096.030

Plasmid sequences. DOI: http://dx.doi.org/10.7554/eLife.18096.031

ELife digest Cells decode genes in two steps. First, they synthesize a molecule similar to DNA, called RNA, which is a complementary copy of the gene. This process, known as transcription, creates an intermediate RNA molecule that is turned into protein in the second step. RNA polymerase is an enzyme that carries out transcription; it separates the...

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor tr...

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor tr...

RNA cleavage by bacterial RNA polymerase (RNAP) has been implicated in transcriptional proofreading and reactivation of arrested transcription elongation complexes but its molecular mechanism is less understood than the mechanism of nucleotide addition, despite both reactions taking place in the same active site. RNAP from the radioresistant bacter...

Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elon...

In this issue of Molecular Cell, Arimbasseri and Maraia (2015) demonstrate that yeast RNA polymerase III integrates inputs from both strands of the DNA template and three dedicated protein subunits to trigger the highly controlled release of the nascent RNA transcript. Copyright © 2015 Elsevier Inc. All rights reserved.

Here we describe a direct fluorescence method that reports real-time occupancies of the pre- and post-translocated state of multisubunit RNA polymerase. In a stopped-flow setup, this method is capable of resolving a single base-pair translocation motion of RNA polymerase in real time. In a conventional spectrofluorometer, this method can be employe...

Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β' bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-β subunit contacts by amino-acid substitutions invariably re...

Two functionally distinct homologous flavoprotein hydroxylases, PgaE and JadH, have been identified as branching points in the biosynthesis of the polyketide antibiotics gaudimycin C and jadomycin A, respectively. These evolutionarily related enzymes are both bifunctional and able to catalyze the same initial reaction, C-12 hydroxylation of the com...

Multisubunit RNA polymerase (RNAP) is the central information-processing enzyme in all cellular life forms, yet its mechanism of translocation along the DNA molecule remains conjectural. Here, we report direct monitoring of bacterial RNAP translocation following the addition of a single nucleotide. Time-resolved measurements demonstrated that trans...

In all organisms, RNA polymerase (RNAP) relies on accessory factors to complete synthesis of long RNAs. These factors increase RNAP processivity by reducing pausing and termination, but their molecular mechanisms remain incompletely understood. We identify the β gate loop as an RNAP element required for antipausing activity of a bacterial virulence...

Membrane pyrophosphatases (PPases), divided into K+-dependent and K+-independent subfamilies, were believed to pump H+ across cell membranes until a recent demonstration that some K+-dependent PPases function as Na+ pumps. Here, we have expressed seven evolutionarily important putative PPases in Escherichia coli and estimated their hydrolytic, Na+...

RfaH is a bacterial antitermination factor whose function depends on a 12‐nt ops element located in the leader regions of several operons; the ops element both induces RNA polymerase (RNAP) pausing and serves as a recognition site for RfaH. During recruitment, RfaH establishes transient contacts with the ops bases in the non‐template DNA strand exp...

Synthesis of long RNA chains by multisubunit RNA polymerases (RNAPs) requires accessory proteins that help RNAP bypass numerous roadblocks it encounters along the way. These “antitermination” proteins switch the elongating RNAP into a processive state, but the molecular mechanism of this phenomena remains unknown. Bacterial antiterminator RfaH is a...

RfaH is a bacterial elongation factor that increases expression of distal genes in several long, horizontally acquired operons. RfaH is recruited to the transcription complex during RNA chain elongation through specific interactions with a DNA element called ops. Following recruitment, RfaH remains bound to RNA polymerase (RNAP) and acts as an anti...

Elongation factors NusG and RfaH evolved from a common ancestor and utilize the same binding site on RNA polymerase (RNAP) to modulate transcription. However, although NusG associates with RNAP transcribing most Escherichia coli genes, RfaH regulates just a few operons containing ops, a DNA sequence that mediates RfaH recruitment. Here, we describe...

Structural studies of antibiotics not only provide a shortcut to medicine allowing for rational structure-based drug design, but may also capture snapshots of dynamic intermediates that become 'frozen' after inhibitor binding. Myxopyronin inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Here we report the structure of dMyx--a desme...

CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II P...

Membrane-bound pyrophosphatase (PPase) is commonly believed to couple pyrophosphate (PPi) hydrolysis to H+ transport across the membrane. Here, we demonstrate that two newly isolated bacterial membrane PPases from the mesophile Methanosarcina mazei (Mm-PPase) and the moderate thermophile Moorella thermoacetica and a previously described PPase from...

RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the alpha-helical coiled-coil C domain, while retaining sequenc...

Efficient transcription of long polycistronic operons in bacteria frequently relies on accessory proteins but their molecular mechanisms remain obscure. RfaH is a cellular elongation factor that acts as a polarity suppressor by increasing RNA polymerase (RNAP) processivity. In this work, we provide evidence that RfaH acts by reducing transcriptiona...

Soluble pyrophosphatases (PPases), which are essential for cell life, comprise two evolutionarily unrelated families (I and II). Prokaryotic genomes generally contain a single PPase gene encoding either family I or family II enzyme. Surprisingly, four Vibrionales species, including the human pathogen Vibrio cholerae, contain PPase genes of both fam...

Membrane-bound pyrophosphatase of the hyperthermophilic bacterium Thermotoga maritima(Tm-PPase), a homologue of H(+)-translocating pyrophosphatase, was expressed in Escherichia coli and isolated as inner membrane vesicles. In contrast to all previously studied H(+)-PPases, both native and recombinant Tm-PPases exhibited an absolute requirement for...

H+-pyrophosphatase (H+-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H+-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine...

The H+ proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-P...

H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of...

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermedia...