Evgeny A Nudler, Ph.D.

Tagetitoxin inhibits RNA polymerase through trapping of the trigger loop
Artsimovitch I.; Svetlov V.; Nemetski S.M.; Epshtein V.; Cardozo T.; Nudler E.
2011 ;286(46):40395-40400, Journal of biological chemistry
Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the beta' subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a 'slow' enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics. 2011 by The American Society for Biochemistry and Molecular Biology, Inc
— id: 142055, year: 2011, vol: 286, page: 40395, stat: Journal Article,

The profound therapeutic effect of exogenous Hsp70 on Alzheimer's type degeneration in olfactory bulbectomized mice
Bobkova N.; Guzhova I.; Margulis B.; Nesterova I.; Medvedinskaya N.; Samokhin A.; Alexandrova I.; Yashin V.; Garbuz D.; Karpov V.; Evgen'ev M.; Nudler E.
2011 ;8:?-?, Neuro-degenerative diseases
Brain deterioration resulting from protein folding diseases, such as the Alzheimer's disease (AD), is one of the leading causes of morbidity and mortality in the aging human population. Heat shock proteins (HSPs) constitute the major cellular quality control system for proteins that mitigates the pathological burden of neurotoxic protein fibrils and aggregates. However, the therapeutic effect of HSPs has not been tested in a relevant setting. Here we report the dramatic neuroprotective effect of recombinant human Hsp70 in the bilateral olfactory bulbectomy (OBE) mouse model. We show that intranasally administered Hsp70 rapidly enters the afflicted brain regions and mitigates multiple AD-like morphological and cognitive abnormalities observed in OBE animals. In particular, it normalized the density of neurons in the hippocampus, which correlated with the diminished accumulation of amyloid b (Ab) peptide. Consistently, Hsp70 also fully protected spatial memory, which remained at the level of control animals for at least eight months following treatment. The long-lasting therapeutic effect of Hsp70 suggests a novel mechanism of action and establishes it as a practical and potent agent for treatment of neurodegenerative diseases associated with abnormal protein biogenesis and cognitive disturbances, such as AD, for which neuroprotective therapy is urgently needed
— id: 136532, year: 2011, vol: 8, page: ?, stat: Journal Article,

Linking RNA Polymerase Backtracking to Genome Instability in E. coli
Dutta, Dipak; Shatalin, Konstantin; Epshtein, Vitaly; Gottesman, Max E; Nudler, Evgeny
2011 Aug 19;146(4):533-543, Cell
Frequent codirectional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Codirectional collisions with backtracked (arrested) ECs lead to DNA double-strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to avoid replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance
— id: 136948, year: 2011, vol: 146, page: 533, stat: Journal Article,

Functional Organization of hsp70 Cluster in Camel (Camelus dromedarius) and Other Mammals
Garbuz, David G; Astakhova, Lubov N; Zatsepina, Olga G; Arkhipova, Irina R; Nudler, Eugene; Evgen'ev, Michael B
2011 ;6(11):e27205-e27205, PLoS ONE
Heat shock protein 70 (Hsp70) is a molecular chaperone providing tolerance to heat and other challenges at the cellular and organismal levels. We sequenced a genomic cluster containing three hsp70 family genes linked with major histocompatibility complex (MHC) class III region from an extremely heat tolerant animal, camel (Camelus dromedarius). Two hsp70 family genes comprising the cluster contain heat shock elements (HSEs), while the third gene lacks HSEs and should not be induced by heat shock. Comparison of the camel hsp70 cluster with the corresponding regions from several mammalian species revealed similar organization of genes forming the cluster. Specifically, the two heat inducible hsp70 genes are arranged in tandem, while the third constitutively expressed hsp70 family member is present in inverted orientation. Comparison of regulatory regions of hsp70 genes from camel and other mammals demonstrates that transcription factor matches with highest significance are located in the highly conserved 250-bp upstream region and correspond to HSEs followed by NF-Y and Sp1 binding sites. The high degree of sequence conservation leaves little room for putative camel-specific regulatory elements. Surprisingly, RT-PCR and 5'/3'-RACE analysis demonstrated that all three hsp70 genes are expressed in camel's muscle and blood cells not only after heat shock, but under normal physiological conditions as well, and may account for tolerance of camel cells to extreme environmental conditions. A high degree of evolutionary conservation observed for the hsp70 cluster always linked with MHC locus in mammals suggests an important role of such organization for coordinated functioning of these vital genes
— id: 142068, year: 2011, vol: 6, page: e27205, stat: Journal Article,

A unified model of transcription elongation: what have we learned from single-molecule experiments?
Maoileidigh, Daibhid O; Tadigotla, Vasisht R; Nudler, Evgeny; Ruckenstein, Andrei E
2011 Mar 2;100(5):1157-1166, Biophysical journal
The transcription of the genetic information encoded in DNA into RNA is performed by RNA polymerase (RNAP), a complex molecular motor, highly conserved across species. Despite remarkable progress in single-molecule techniques revealing important mechanistic details of transcription elongation (TE) with up to base-pair resolution, some of the results and interpretations of these studies are difficult to reconcile, and have not yet led to a minimal unified picture of transcription. We propose a simple model that accounts quantitatively for many of the experimental observations. This model belongs to the class of isothermal ratchet models of TE involving the thermally driven stochastic backward and forward motion (backtracking and forward tracking) of RNAP along DNA between single-nucleotide incorporation events. We uncover two essential features for the success of the model. The first is an intermediate state separating the productive elongation pathway from nonelongating backtracked states. The rates of entering and exiting this intermediate state modulate pausing by RNAP. The second crucial ingredient of the model is the cotranscriptional folding of the RNA transcript, sterically inhibiting the extent of backtracking. This model resolves several apparent differences between single-molecule studies and provides a framework for future work on TE
— id: 134116, year: 2011, vol: 100, page: 1157, stat: Journal Article,

H2S: a universal defense against antibiotics in bacteria
Shatalin, Konstantin; Shatalina, Elena; Mironov, Alexander; Nudler, Evgeny
2011 Nov 18;334(6058):986-990, Science
Many prokaryotic species generate hydrogen sulfide (H(2)S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine beta-synthase, cystathionine gamma-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H(2)S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H(2)S suppresses this effect. Moreover, in bacteria that normally produce H(2)S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics
— id: 141709, year: 2011, vol: 334, page: 986, stat: Journal Article,

Clamping the clamp of RNA polymerase
Svetlov, Vladimir; Nudler, Evgeny
2011 Apr 6;30(7):1190-1191, EMBO journal
— id: 130311, year: 2011, vol: 30, page: 1190, stat: Journal Article,

An allosteric mechanism of Rho-dependent transcription termination
Epshtein, Vitaly; Dutta, Dipak; Wade, Joseph; Nudler, Evgeny
2010 Jan 14;463(7278):245-249, Nature
Rho is the essential RNA helicase that sets the borders between transcription units and adjusts transcriptional yield to translational needs in bacteria. Although Rho was the first termination factor to be discovered, the actual mechanism by which it reaches and disrupts the elongation complex (EC) is unknown. Here we show that the termination-committed Rho molecule associates with RNA polymerase (RNAP) throughout the transcription cycle; that is, it does not require the nascent transcript for initial binding. Moreover, the formation of the RNAP-Rho complex is crucial for termination. We show further that Rho-dependent termination is a two-step process that involves rapid EC inactivation (trap) and a relatively slow dissociation. Inactivation is the critical rate-limiting step that establishes the position of the termination site. The trap mechanism depends on the allosterically induced rearrangement of the RNAP catalytic centre by means of the evolutionarily conserved mobile trigger-loop domain, which is also required for EC dissociation. The key structural and functional similarities, which we found between Rho-dependent and intrinsic (Rho-independent) termination pathways, argue that the allosteric mechanism of termination is general and likely to be preserved for all cellular RNAPs throughout evolution
— id: 106244, year: 2010, vol: 463, page: 245, stat: Journal Article,

Cooperation between translating ribosomes and RNA polymerase in transcription elongation
Proshkin, Sergey; Rahmouni, A Rachid; Mironov, Alexander; Nudler, Evgeny
2010 Apr 23;328(5977):504-508, Science
During transcription of protein-coding genes, bacterial RNA polymerase (RNAP) is closely followed by a ribosome that translates the newly synthesized transcript. Our in vivo measurements show that the overall elongation rate of transcription is tightly controlled by the rate of translation. Acceleration and deceleration of a ribosome result in corresponding changes in the speed of RNAP. Moreover, we found an inverse correlation between the number of rare codons in a gene, which delay ribosome progression, and the rate of transcription. The stimulating effect of a ribosome on RNAP is achieved by preventing its spontaneous backtracking, which enhances the pace and also facilitates readthrough of roadblocks in vivo. Such a cooperative mechanism ensures that the transcriptional yield is always adjusted to translational needs at different genes and under various growth conditions
— id: 109517, year: 2010, vol: 328, page: 504, stat: Journal Article,

Transcription inactivation through local refolding of the RNA polymerase structure
Belogurov, Georgiy A; Vassylyeva, Marina N; Sevostyanova, Anastasiya; Appleman, James R; Xiang, Alan X; Lira, Ricardo; Webber, Stephen E; Klyuyev, Sergiy; Nudler, Evgeny; Artsimovitch, Irina; Vassylyev, Dmitry G
2009 Jan 15;457(7227):332-335, Nature
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 desmethyl derivative of myxopyronin B--complexed with a Thermus thermophilus RNAP holoenzyme. The antibiotic binds to a pocket deep inside the RNAP clamp head domain, which interacts with the DNA template in the transcription bubble. Notably, binding of dMyx stabilizes refolding of the beta'-subunit switch-2 segment, resulting in a configuration that might indirectly compromise binding to, or directly clash with, the melted template DNA strand. Consistently, footprinting data show that the antibiotic binding does not prevent nucleation of the promoter DNA melting but instead blocks its propagation towards the active site. Myxopyronins are thus, to our knowledge, a first structurally characterized class of antibiotics that target formation of the pre-catalytic transcription initiation complex-the decisive step in gene expression control. Notably, mutations designed in switch-2 mimic the dMyx effects on promoter complexes in the absence of antibiotic. Overall, our results indicate a plausible mechanism of the dMyx action and a stepwise pathway of open complex formation in which core enzyme mediates the final stage of DNA melting near the transcription start site, and that switch-2 might act as a molecular checkpoint for DNA loading in response to regulatory signals or antibiotics. The universally conserved switch-2 may have the same role in all multisubunit RNAPs
— id: 108810, year: 2009, vol: 457, page: 332, stat: Journal Article,

Endogenous nitric oxide protects bacteria against a wide spectrum of antibiotics
Gusarov, Ivan; Shatalin, Konstantin; Starodubtseva, Marina; Nudler, Evgeny
2009 Sep 11;325(5946):1380-1384, Science
Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms. NO-mediated resistance is achieved through both the chemical modification of toxic compounds and the alleviation of the oxidative stress imposed by many antibiotics. Our results suggest that the inhibition of NOS activity may increase the effectiveness of antimicrobial therapy
— id: 102402, year: 2009, vol: 325, page: 1380, stat: Journal Article,

Transcriptional approaches to riboswitch studies
Mironov, Alexander; Epshtein, Vitaly; Nudler, Evgeny
2009 ;540:39-51, Methods in molecular biology
Natural RNA sensors of small molecules (a.k.a. riboswitches) regulate numerous metabolic genes. In bacteria, these RNA elements control transcription termination and translation initiation by changing the folding pathway of nascent RNA upon direct binding of a metabolite. To identify and study riboswitches we used in vitro reconstituted solid-phase transcription elongation/termination system. This approach allows for direct monitoring of ligand binding and riboswitch functioning, establishing the working concentration of a ligand as a function of RNA polymerase speed, and also probing RNA structure of the riboswitch. Using this system we have been able to identify and characterize first several riboswitches including those involved in vitamin biosynthesis and sulfur metabolism. The system can be utilized to facilitate biochemical studies of riboswitches in general, i.e., to simplify analysis of riboswitches that are not necessarily involved in transcriptional control
— id: 98005, year: 2009, vol: 540, page: 39, stat: Journal Article,

RNA polymerase active center: the molecular engine of transcription
Nudler, Evgeny
2009 ;78:335-361, Annual review of biochemistry
RNA polymerase (RNAP) is a complex molecular machine that governs gene expression and its regulation in all cellular organisms. To accomplish its function of accurately producing a full-length RNA copy of a gene, RNAP performs a plethora of chemical reactions and undergoes multiple conformational changes in response to cellular conditions. At the heart of this machine is the active center, the engine, which is composed of distinct fixed and moving parts that serve as the ultimate acceptor of regulatory signals and as the target of inhibitory drugs. Recent advances in the structural and biochemical characterization of RNAP explain the active center at the atomic level and enable new approaches to understanding the entire transcription mechanism, its exceptional fidelity and control
— id: 99246, year: 2009, vol: 78, page: 335, stat: Journal Article,

Isolation and characterization of the heat shock RNA 1
Shamovsky, Ilya; Nudler, Evgeny
2009 ;540:265-279, Methods in molecular biology
The heat shock (HS) response is the major cellular defense mechanism against acute exposure to environmental stresses. The hallmark of the HS response, which is conserved in all eukaryotes, is the rapid and massive induction of expression of a set of cytoprotective genes. Most of the induction occurs at the level of transcription. The master regulator, heat shock transcription factor (HSF, or HSF1 in vertebrates), is responsible for the induction of HS gene transcription in response to elevated temperature. Under normal conditions HSF is present in the cell as an inactive monomer. During HS, HSF trimerizes and binds to a consensus sequence in the promoter of HS genes, stimulating their transcription by up to 200-fold. We have shown that a large, noncoding RNA, HSR1, and the translation elongation factor eEF1A form a complex with HSF during HS and are required for its activation
— id: 98006, year: 2009, vol: 540, page: 265, stat: Journal Article,

Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response
Shen, XH; Banga, S; Liu, YC; Xu, L; Gao, P; Shamovsky, I; Nudler, E; Luo, ZQ
2009 JUN ;11(6):911-926, Cellular microbiology
The Legionella pneumophila Dot/Icm type IV secretion system is essential for the biogenesis of a phagosome that supports bacterial multiplication, most likely via the functions of its protein substrates. Recent studies indicate that fundamental cellular processes, such as vesicle trafficking, stress response, autophagy and cell death, are modulated by these effectors. However, how each translocated protein contributes to the modulation of these pathways is largely unknown. In a screen to search substrates of the Dot/Icm transporter that can cause host cell death, we identified a gene whose product is lethal to yeast and mammalian cells. We demonstrate that this protein, called SidI, is a substrate of the Dot/Icm type IV protein transporter that targets the host protein translation process. Our results indicate that SidI specifically interacts with eEF1A and eEF1B gamma, two components of the eukaryotic protein translation elongation machinery and such interactions leads to inhibition of host protein synthesis. Furthermore, we have isolated two SidI substitution mutants that retain the target binding activity but have lost toxicity to eukaryotic cells, suggesting potential biochemical effect of SidI on eEF1A and eEF1B gamma. We also show that infection by L. pneumophila leads to eEF1A-mediated activation of the heat shock regulatory protein HSF1 in a virulence-dependent manner and deletion of sidI affects such activation. Moreover, similar response occurred in cells transiently transfected to express SidI. Thus, inhibition of host protein synthesis by specific effectors contributes to the induction of stress response in L. pneumophila-infected cells
— id: 98998, year: 2009, vol: 11, page: 911, stat: Journal Article,

Macromolecular micromovements: how RNA polymerase translocates
Svetlov, Vladimir; Nudler, Evgeny
2009 Dec;19(6):701-707, Current opinion in structural biology
Multi-subunit DNA-dependent RNA polymerases synthesize RNA molecules thousands of nucleotides long. The reiterative reaction of nucleotide condensation occurs at rates of tens of nucleotides per second, invariably linked to the translocation of the enzyme along the DNA template, or threading of the DNA and the nascent RNA molecule through the enzyme. Reiteration of the nucleotide addition/translocation cycle without dissociation from the DNA and RNA requires both isomorphic and metamorphic conformational flexibility of a magnitude substantial enough to accommodate the requisite molecular motions. Here we review some of the more recently acquired insights into the structural flexibility and morphic fluctuations of RNA polymerases and their mechanistic implications
— id: 105503, year: 2009, vol: 19, page: 701, stat: Journal Article,

RNA polymerase: the vehicle of transcription
Borukhov, Sergei; Nudler, Evgeny
2008 Mar;16(3):126-134, Trends in microbiology
RNA polymerase (RNAP) is the principal enzyme of gene expression and regulation for all three divisions of life: Eukaryota, Archaea and Bacteria. Recent progress in the structural and biochemical characterization of RNAP illuminates this enzyme as a flexible, multifunctional molecular machine. During each step of the transcription cycle, RNAP undergoes elaborate conformational changes. As many fundamental and previously mysterious aspects of how RNAP works begin to be understood, this enzyme reveals intriguing similarities to man-made engineered devices. These resemblances can be found in the mechanics of RNAP-DNA complex formation, in RNA chain initiation and in the elongation processes. Here we highlight recent advances in understanding RNAP function and regulation
— id: 76132, year: 2008, vol: 16, page: 126, stat: Journal Article,

Termination factor Rho and its cofactors NusA and NusG silence foreign DNA in E. coli
Cardinale, Christopher J; Washburn, Robert S; Tadigotla, Vasisht R; Brown, Lewis M; Gottesman, Max E; Nudler, Evgeny
2008 May 16;320(5878):935-938, Science
Transcription of the bacterial genome by the RNA polymerase must terminate at specific points. Transcription can be terminated by Rho factor, an essential protein in enterobacteria. We used the antibiotic bicyclomycin, which inhibits Rho, to assess its role on a genome-wide scale. Rho is revealed as a global regulator of gene expression that matches Escherichia coli transcription to translational needs. We also found that genes in E. coli that are most repressed by Rho are prophages and other horizontally acquired portions of the genome. Elimination of these foreign DNA elements increases resistance to bicyclomycin. Although rho remains essential, such reduced-genome bacteria no longer require Rho cofactors NusA and NusG. Deletion of the cryptic rac prophage in wild-type E. coli increases bicyclomycin resistance and permits deletion of nusG. Thus, Rho termination, supported by NusA and NusG, is required to suppress the toxic activity of foreign genes
— id: 78869, year: 2008, vol: 320, page: 935, stat: Journal Article,

Bacterial NO-synthases operate without a dedicated redox partner
Gusarov, Ivan; Starodubtseva, Marina; Wang, Zhi-Qiang; McQuade, Lindsey; Lippard, Stephen J; Stuehr, Dennis J; Nudler, Evgeny
2008 May 9;283(19):13140-13147, Journal of biological chemistry
Bacterial NO-synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These 'promiscuous' bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in E. coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO-donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO-donors for research and clinical needs
— id: 76130, year: 2008, vol: 283, page: 13140, stat: Journal Article,

New insights into the mechanism of heat shock response activation
Shamovsky, I; Nudler, E
2008 Mar;65(6):855-861, Cellular & molecular life sciences: CMLS
Heat shock (HS) response is a universal mechanism of protection against adverse environmental conditions. It is manifested mainly by rapid and robust induction of molecular chaperones and other cytoprotective proteins. In higher eukaryotes the activation of the HS response is mediated by a master regulator, heat shock transcription factor 1 (HSF1). Here we outline recent progress in understanding the early steps in HSF1 activation by heat in the context of existing models of HSF1 regulation
— id: 79154, year: 2008, vol: 65, page: 855, stat: Journal Article,

Modular RNA heats up
Shamovsky, Ilya; Nudler, Evgeny
2008 Feb 29;29(4):415-417, Molecular cell
In this issue of Molecular Cell, Mariner et al. (2008) demonstrate that Alu RNA from a human SINE represses RNA polymerase II transcription during heat shock. This noncoding RNA is the first example of a 'protein-like' transcription factor with a distinct modular architecture
— id: 76131, year: 2008, vol: 29, page: 415, stat: Journal Article,

Bacillus anthracis-derived nitric oxide is essential for pathogen virulence and survival in macrophages
Shatalin, Konstantin; Gusarov, Ivan; Avetissova, Ekaterina; Shatalina, Yelena; McQuade, Lindsey E; Lippard, Stephen J; Nudler, Evgeny
2008 Jan 22;105(3):1009-1013, Proceedings of the National Academy of Sciences of the United States of America
Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases
— id: 75858, year: 2008, vol: 105, page: 1009, stat: Journal Article,

Jamming the ratchet of transcription
Svetlov, V; Nudler, E
2008 AUG ;15(8):777-779, Nature structural & molecular biology
The exact mechanism by which cellular RNA polymerases translocate and maintain exceptionally high fidelity during transcription remains an important unresolved issue. Two recent structural studies of yeast RNA polymerase II in complex with its potent inhibitor, the fungal toxin alpha-amanitin, address this matter by describing crucial and details about the dynamic organization of the enzyme catalytic center
— id: 86821, year: 2008, vol: 15, page: 777, stat: Journal Article,

Riboswitch-dependent gene regulation and its evolution in the plant kingdom
Bocobza, Samuel; Adato, Avital; Mandel, Tali; Shapira, Michal; Nudler, Evgeny; Aharoni, Asaph
2007 Nov 15;21(22):2874-2879, Genes & development
Riboswitches are natural RNA sensors that affect gene control via their capacity to bind small molecules. Their prevalence in higher eukaryotes is unclear. We discovered a post-transcriptional mechanism in plants that uses a riboswitch to control a metabolic feedback loop through differential processing of the precursor RNA 3' terminus. When cellular thiamin pyrophosphate (TPP) levels rise, metabolite sensing by the riboswitch located in TPP biosynthesis genes directs formation of an unstable splicing product, and consequently TPP levels drop. When transformed in plants, engineered TPP riboswitches can act autonomously to modulate gene expression. In an evolutionary perspective, a TPP riboswitch is also present in ancient plant taxa, suggesting that this mechanism is active since vascular plants emerged 400 million years ago
— id: 76133, year: 2007, vol: 21, page: 2874, stat: Journal Article,

An allosteric path to transcription termination
Epshtein, Vitaly; Cardinale, Christopher J; Ruckenstein, Andrei E; Borukhov, Sergei; Nudler, Evgeny
2007 Dec 28;28(6):991-1001, Molecular cell
Transcription termination signals in bacteria occur in RNA as a strong hairpin followed by a stretch of U residues at the 3' terminus. To release the transcript, RNA polymerase (RNAP) is thought to translocate forward without RNA synthesis. Here we provide genetic and biochemical evidence supporting an alternative model in which extensive conformational changes across the enzyme lead to termination without forward translocation. In this model, flexible parts of the RNA exit channel (zipper, flap, and zinc finger) assist the initial step of hairpin folding (nucleation). The hairpin then invades the RNAP main channel, causing RNA:DNA hybrid melting, structural changes of the catalytic site, and DNA-clamp opening induced by interaction with the G(trigger)-loop. Our results envision the elongation complex as a flexible structure, not a rigid body, and establish basic principles of the termination pathway that are likely to be universal in prokaryotic and eukaryotic systems
— id: 75853, year: 2007, vol: 28, page: 991, stat: Journal Article,

Assessment of nitric oxide signals by triiodide chemiluminescence
Hausladen, Alfred; Rafikov, Ruslan; Angelo, Michael; Singel, David J; Nudler, Evgeny; Stamler, Jonathan S
2007 Feb 13;104(7):2157-2162, Proceedings of the National Academy of Sciences of the United States of America
Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures
— id: 76134, year: 2007, vol: 104, page: 2157, stat: Journal Article,

Adaptive mutations in RNA-based regulatory mechanisms: Computational and experimental investigations
Barash, D; Sikorski, J; Perry, EB; Nevo, E; Nudler, E
2006 NOV-DEC ;52(3-4):263-279, Israel journal of ecology & evolution
Recent discoveries of RNA-based regulatory mechanisms have prompted substantial interest in how they formed and the extent to which varying environmental conditions have influenced their evolution. One class of RNA-based regulatory mechanism that has been found in bacteria is the riboswitch, regulating the biosynthesis of certain vitamins by an RNA genetic control clement that senses small molecules and responds with a structural change that affects transcription termination or translation initiation without the participation of proteins. By taking the thiamin pyrophosphate (TPP)-riboswitch in Bacillus subtilis as a model system, we wish to examine whether beneficial mutations may exist at the level of RNA that will cause an improvement in organism fitness. By computationally analyzing the difference in primary and secondary structure of the B. subtilis TPP-riboswitch collected from the xeric 'African' south-facing slope (SFS) vs. the mesic, 'European', north-facing slope (NFS) in 'Evolution Canyon' III at Nahal Shaharut, southern Israel, we wish to experimentally study the environmental effect on transcription termination in these RNA-based regulatory mechanisms that are believed to be of ancient origin in the evolutionary time scale. Computational results, so far, indicate that specific mutations affect the riboswitch conformation by causing a global rearrangement. We would like to check whether such mutations could be adaptive mutations that may have a beneficial fitness effect, taking the TPP-riboswitch as a model system at the micro-scale. Empirical results so far indicate that in the promoter region of the TPP-riboswitch, all mutations increase nucleotide GC content in the xeric SFS, whereas in the mesic NFS they increase AT content. Preliminary examination of termination efficiency of strains found exclusively on one slope or the other, reveal increased termination efficiency in the presence of TPP and at more moderate temperatures, but only a suggestion of greater termination efficiency from strains found on both slopes. We expect that further results will shed light on the mutational differences of TPP-riboswitch sequences found on opposite slopes of 'Evolution Canyon' III at Nahal Shaharut, potentially leading to interesting discoveries that relate to the topic of adaptive, nonrandom mutations
— id: 75200, year: 2006, vol: 52, page: 263, stat: Journal Article,

Transcription regulatory elements are punctuation marks for DNA replication
Mirkin, Ekaterina V; Castro Roa, Daniel; Nudler, Evgeny; Mirkin, Sergei M
2006 May 9;103(19):7276-7281, Proceedings of the National Academy of Sciences of the United States of America
Collisions between DNA replication and transcription significantly affect genome organization, regulation, and stability. Previous studies have described collisions between replication forks and elongating RNA polymerases. Although replication collisions with the transcription-initiation or -termination complexes are potentially even more important because most genes are not actively transcribed during DNA replication, their existence and mechanisms remained unproven. To address this matter, we have designed a bacterial promoter that binds RNA polymerase and maintains it in the initiating mode by precluding the transition into the elongation mode. By using electrophoretic analysis of replication intermediates, we have found that this steadfast transcription-initiation complex inhibits replication fork progression in an orientation-dependent manner during head-on collisions. Transcription terminators also appeared to attenuate DNA replication, but in the opposite, codirectional orientation. Thus, transcription regulatory signals may serve as 'punctuation marks' for DNA replication in vivo
— id: 76136, year: 2006, vol: 103, page: 7276, stat: Journal Article,

Flipping riboswitches
Nudler, Evgeny
2006 Jul 14;126(1):19-22, Cell
Riboswitches are common cis-acting regulatory elements in bacteria. They are made of nascent RNA that changes its conformation in response to direct binding of cognate metabolites. The publication of five high-resolution crystal structures provides a comprehensive view of how riboswitches sense their ligands and points to new challenges in this emerging field
— id: 67853, year: 2006, vol: 126, page: 19, stat: Journal Article,

RNA-mediated response to heat shock in mammalian cells
Shamovsky, Ilya; Ivannikov, Maxim; Kandel, Eugene S; Gershon, David; Nudler, Evgeny
2006 Mar 23;440(7083):556-560, Nature
The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation
— id: 64139, year: 2006, vol: 440, page: 556, stat: Journal Article,

Gene control by large noncoding RNAs
Shamovsky, Ilya; Nudler, Evgeny
2006 Oct 3;2006(355):pe40-pe40, Science's STKE
Large noncoding RNAs (lncRNAs) have emerged as key players in regulating various fundamental cellular processes. Recent reports identify a functional lncRNA, Evf-2, that operates during development to control the expression of specific homeodomain proteins, and they provide important insights into the mechanism of cooperation between a newly discovered nuclear receptor co-repressor protein (SLIRP) and steroid receptor activator RNA. Evf-2 is the first example of lncRNA directly involved in organogenesis in vertebrates
— id: 69081, year: 2006, vol: 2006, page: pe40, stat: Journal Article,

Thermodynamic and kinetic modeling of transcriptional pausing
Tadigotla, Vasisht R; O Maoileidigh, Daibhid; Sengupta, Anirvan M; Epshtein, Vitaly; Ebright, Richard H; Nudler, Evgeny; Ruckenstein, Andrei E
2006 Mar 21;103(12):4439-4444, Proceedings of the National Academy of Sciences of the United States of America
We present a statistical mechanics approach for the prediction of backtracked pauses in bacterial transcription elongation derived from structural models of the transcription elongation complex (EC). Our algorithm is based on the thermodynamic stability of the EC along the DNA template calculated from the sequence-dependent free energy of DNA-DNA, DNA-RNA, and RNA-RNA base pairing associated with (i) the translocational and size fluctuations of the transcription bubble; (ii) changes in the associated DNA-RNA hybrid; and (iii) changes in the cotranscriptional RNA secondary structure upstream of the RNA exit channel. The calculations involve no adjustable parameters except for a cutoff used to discriminate paused from nonpaused complexes. When applied to 100 experimental pauses in transcription elongation by Escherichia coli RNA polymerase on 10 DNA templates, the approach produces statistically significant results. We also present a kinetic model for the rate of recovery of backtracked paused complexes. A crucial ingredient of our model is the incorporation of kinetic barriers to backtracking resulting from steric clashes of EC with the cotranscriptionally generated RNA secondary structure, an aspect not included explicitly in previous attempts at modeling the transcription elongation process
— id: 76137, year: 2006, vol: 103, page: 4439, stat: Journal Article,

Extensive functional overlap between sigma factors in Escherichia coli
Wade, Joseph T; Roa, Daniel Castro; Grainger, David C; Hurd, Douglas; Busby, Stephen J W; Struhl, Kevin; Nudler, Evgeny
2006 Sep;13(9):806-814, Nature structural & molecular biology
Bacterial core RNA polymerase (RNAP) must associate with a sigma factor to recognize promoter sequences. Escherichia coli encodes seven sigma factors, each believed to be specific for a largely distinct subset of promoters. Using microarrays representing the entire E. coli genome, we identify 87 in vivo targets of sigma32, the heat-shock sigma factor, and estimate that there are 120-150 sigma32 promoters in total. Unexpectedly, 25% of these sigma32 targets are located within coding regions, suggesting novel regulatory roles for sigma32. The majority of sigma32 promoter targets overlap with those of sigma70, the housekeeping sigma factor. Furthermore, their DNA sequence motifs are often interdigitated, with RNAPsigma70 and RNAPsigma32 initiating transcription in vitro with similar efficiency and from identical positions. SigmaE-regulated promoters also overlap extensively with those for sigma70. These results suggest that extensive functional overlap between sigma factors is an important phenomenon
— id: 76135, year: 2006, vol: 13, page: 806, stat: Journal Article,

A ratchet mechanism of transcription elongation and its control
Bar-Nahum, Gil; Epshtein, Vitaly; Ruckenstein, Andrei E; Rafikov, Ruslan; Mustaev, Arkady; Nudler, Evgeny
2005 Jan 28;120(2):183-193, Cell
RNA chain elongation is a highly processive and accurate process that is finely regulated by numerous intrinsic and extrinsic signals. Here we describe a general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs such as pauses, terminators, and elongation factors. We show that E.coli RNAP moves by a complex Brownian ratchet mechanism, which acts prior to phosphodiester bond formation. The incoming substrate and the flexible F bridge domain of the catalytic center serve as two separate ratchet devices that function in concert to drive forward translocation. The adjacent G loop domain controls F bridge motion, thus keeping the proper balance between productive and inactive states of the elongation complex. This balance is critical for cell viability since it determines the rate, processivity, and fidelity of transcription
— id: 48115, year: 2005, vol: 120, page: 183, stat: Journal Article,

NO-mediated cytoprotection: instant adaptation to oxidative stress in bacteria
Gusarov, Ivan; Nudler, Evgeny
2005 Sep 27;102(39):13855-13860, Proceedings of the National Academy of Sciences of the United States of America
Numerous sophisticated systems have been described that protect bacteria from increased levels of reactive oxygen species. Although indispensable during prolonged oxidative stress, these response systems depend on newly synthesized proteins, and are hence both time and energy consuming. Here, we describe an 'express' cytoprotective system in Bacillus subtilis which depends on nitric oxide (NO). We show that NO immediately protects bacterial cells from reactive oxygen species by two independent mechanisms. NO transiently suppresses the enzymatic reduction of free cysteine that fuels the damaging Fenton reaction. In addition, NO directly reactivates catalase, a major antioxidant enzyme that has been inhibited in vivo by endogenous cysteine. Our data also reveal a critical role for bacterial NO-synthase in adaptation to oxidative stress associated with fast metabolic changes, and suggest a possible role for NO in defending pathogens against immune oxidative attack
— id: 76138, year: 2005, vol: 102, page: 13855, stat: Journal Article,

Genome review
Nudler, E
2004 MAR ;290(3):10-10, Scientific american
— id: 42501, year: 2004, vol: 290, page: 10, stat: Journal Article,

The riboswitch control of bacterial metabolism
Nudler, Evgeny; Mironov, Alexander S
2004 Jan;29(1):11-17, Trends in biochemical sciences
Aptamers are artificial nucleic acids that selectively bind small molecules. In the past two years, it has become clear that nature has already devised its own aptamers that play important regulatory roles. RNA sensors have been discovered in both Gram-positive and Gram-negative bacteria that function as molecular switches in response to direct binding of structurally diverse metabolites. These natural RNA aptamers, called 'riboswitches', are imbedded in the leader sequences of numerous metabolic genes. Riboswitches are able to repress or activate their cognate genes at both transcriptional and translational levels. Here, we summarize the recent progress in the identification and characterization of riboswitches and discuss their evolution and distribution
— id: 42616, year: 2004, vol: 29, page: 11, stat: Journal Article,

Control of plasma nitric oxide bioactivity by perfluorocarbons: physiological mechanisms and clinical implications
Rafikova, Olga; Sokolova, Elena; Rafikov, Ruslan; Nudler, Evgeny
2004 Dec 7;110(23):3573-3580, Circulation
BACKGROUND: Perfluorocarbons (PFCs) are promising blood substitutes because of their chemical inertness and unparalleled ability to transport and upload O2 and CO2. Here, we report that PFC emulsions also efficiently absorb and transport nitric oxide (NO). METHODS AND RESULTS: Accumulation of NO and O2 in PFC micelles results in rapid NO oxidation and generation of reactive NO(x) species. Such micellar catalysis of NO oxidation leads to formation of vasoactive S-nitrosothiols (RSNO) in vitro and in vivo as detected electrochemically. The efficiency of PFC-mediated S-nitrosation depends on the amount of PFC in aqueous solution. The optimal PFC concentration that produced the maximum level of RSNO was approximately 1% (vol/vol). Larger PFC amounts were progressively less efficient in generating RSNO and functioned simply as NO sink. These results explain the characteristic hemodynamic effects of PFCs. Intravenous bolus application of PFC (0.14 g/kg, approximately 1% vol/vol) to Wistar-Kyoto rats decreased mean arterial pressure significantly (-10 mm Hg over 40 minutes). PFC-induced hypotension could be further stimulated (-17 mm Hg over 140 minutes) by exogenous thiols (cysteine and glutathione). In contrast, a larger amount of PFC (1 g/kg, approximately 7% vol/vol) exhibited a strong hypertensive effect (11 mm Hg over 40 minutes). CONCLUSIONS: The present study reveals a physiologically significant pool of endogenous plasma NO and underscores the crucial role of the circulating hydrophobic phase in modulating its bioactivity. The results also establish PFC as a conceptually new pharmacological tool for various cardiovascular complications associated with NO imbalance
— id: 48116, year: 2004, vol: 110, page: 3573, stat: Journal Article,

RNA polymerase holoenzyme: structure, function and biological implications
Borukhov, Sergei; Nudler, Evgeny
2003 Apr;6(2):93-100, Current opinion in microbiology
The past three years have marked the breakthrough in our understanding of the structural and functional organization of RNA polymerase. The latest major advance was the high-resolution structures of bacterial RNA polymerase holoenzyme and the holoenzyme in complex with promoter DNA. Together with an array of genetic, biochemical and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of the transcription cycle. They include the binding of sigma factor to the core enzyme, and the recognition of promoter sequences and DNA melting by holoenzyme, transcription initiation and promoter clearance
— id: 48118, year: 2003, vol: 6, page: 93, stat: Journal Article,

The riboswitch-mediated control of sulfur metabolism in bacteria
Epshtein, Vitaly; Mironov, Alexander S; Nudler, Evgeny
2003 Apr 29;100(9):5052-5056, Proceedings of the National Academy of Sciences of the United States of America
Many operons in Gram-positive bacteria that are involved in methionine (Met) and cysteine (Cys) biosynthesis possess an evolutionarily conserved regulatory leader sequence (S-box) that positively controls these genes in response to methionine starvation. Here, we demonstrate that a feed-back regulation mechanism utilizes S-adenosyl-methionine as an effector. S-adenosyl-methionine directly and specifically binds to the nascent S-box RNA, causing an intrinsic terminator to form and interrupt transcription prematurely. The S-box leader RNA thus expands the family of newly discovered riboswitches, i.e., natural regulatory RNA aptamers that seem to sense small molecules ranging from amino acid derivatives to vitamins
— id: 48120, year: 2003, vol: 100, page: 5052, stat: Journal Article,

Cooperation between RNA polymerase molecules in transcription elongation
Epshtein, Vitaly; Nudler, Evgeny
2003 May 2;300(5620):801-805, Science
Transcription elongation is responsible for rapid synthesis of RNA chains of thousands of nucleotides in vivo. In contrast, a single round of transcription performed in vitro is frequently interrupted by pauses and arrests that drastically reduce the elongation rate and the yield of the full-length transcript. Here we demonstrate that most transcriptional delays disappear if more than one RNA polymerase (RNAP) molecule initiates from the same promoter. Anti-arrest and anti-pause effects of trailing RNAP are due to forward translocation of leading (backtracked) complexes. Such cooperation between RNAP molecules links the rate of elongation to the rate of initiation and explains why elongation is still fast and processive in vivo even without anti-arrest factors
— id: 48119, year: 2003, vol: 300, page: 801, stat: Journal Article,

Transcription through the roadblocks: the role of RNA polymerase cooperation
Epshtein, Vitaly; Toulme, Francine; Rahmouni, A Rachid; Borukhov, Sergei; Nudler, Evgeny
2003 Sep 15;22(18):4719-4727, EMBO journal
During transcription, cellular RNA polymerases (RNAP) have to deal with numerous potential roadblocks imposed by various DNA binding proteins. Many such proteins partially or completely interrupt a single round of RNA chain elongation in vitro. Here we demonstrate that Escherichia coli RNAP can effectively read through the site-specific DNA-binding proteins in vitro and in vivo if more than one RNAP molecule is allowed to initiate from the same promoter. The anti-roadblock activity of the trailing RNAP does not require transcript cleavage activity but relies on forward translocation of roadblocked complexes. These results support a cooperation model of transcription whereby RNAP molecules behave as 'partners' helping one another to traverse intrinsic and extrinsic obstacles
— id: 48117, year: 2003, vol: 22, page: 4719, stat: Journal Article,

Characterization of protein-nucleic acid interactions that are required for transcription processivity
Nudler, Evgeny; Avetissova, Ekaterina; Korzheva, Nataliya; Mustaev, Arkady
2003 ;371(5):179-190, Methods in enzymology
— id: 46281, year: 2003, vol: 371, page: 179, stat: Journal Article,

Analysis of the intrinsic transcription termination mechanism and its control
Nudler, Evgeny; Gusarov, Ivan
2003 ;371(5):369-382, Methods in enzymology
— id: 46280, year: 2003, vol: 371, page: 369, stat: Journal Article,

Methods of walking with the RNA polymerase
Nudler, Evgeny; Gusarov, Ivan; Bar-Nahum, Gil
2003 ;371(5):160-169, Methods in enzymology
— id: 46282, year: 2003, vol: 371, page: 160, stat: Journal Article,

Template switching by RNA polymerase II in vivo. Evidence and implications from a retroviral system
Kandel, Eugene S; Nudler, Evgeny
2002 Dec;10(6):1495-1502, Molecular cell
Transfection of retrovirus packaging cells with linear DNA from a retroviral vector missing the 3' long terminal repeat (3' LTR) results in production of infectious virus. Analysis of the newly formed proviruses indicates that restoration of the 3' LTR sequences necessary for reverse transcription and integration occurred due to end-to-end template switching by mammalian RNA polymerase II (RNAP II) in the packaging cells. These observations argue that RNAP II can utilize double-strand breaks and gaps in DNA to generate 'recombinant' transcripts in vivo and suggest a mechanism for mutation and recombination of retroviruses
— id: 48121, year: 2002, vol: 10, page: 1495, stat: Journal Article,

Sensing small molecules by nascent RNA: a mechanism to control transcription in bacteria
Mironov, Alexander S; Gusarov, Ivan; Rafikov, Ruslan; Lopez, Lubov Errais; Shatalin, Konstantin; Kreneva, Rimma A; Perumov, Daniel A; Nudler, Evgeny
2002 Nov 27;111(5):747-756, Cell
Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well
— id: 48122, year: 2002, vol: 111, page: 747, stat: Journal Article,

Transcription termination and anti-termination in E. coli
Nudler, Evgeny; Gottesman, Max E
2002 Aug;7(8):755-768, Genes to cells
Transcription termination in Escherichia coli is controlled by many factors. The sequence of the DNA template, the structure of the transcript, and the actions of auxiliary proteins all play a role in determining the efficiency of the process. Termination is regulated and can be enhanced or suppressed by host and phage proteins. This complex reaction is rapidly yielding to biochemical and structural analysis of the interacting factors. Below we review and attempt to unify into basic principles the remarkable recent progress in understanding transcription termination and anti-termination
— id: 48123, year: 2002, vol: 7, page: 755, stat: Journal Article,

Catalysis of S-nitrosothiols formation by serum albumin: the mechanism and implication in vascular control
Rafikova, Olga; Rafikov, Ruslan; Nudler, Evgeny
2002 Apr 30;99(9):5913-5918, Proceedings of the National Academy of Sciences of the United States of America
Nitric oxide (NO(.)) is a short-lived physiological messenger. Its various biological activities can be preserved in a more stable form of S-nitrosothiols (RS-NO). Here we demonstrate that at physiological NO(.) concentrations, plasma albumin becomes saturated with NO(.) and accelerates formation of low-molecular-weight (LMW) RS-NO in vitro and in vivo. The mechanism involves micellar catalysis of NO(.) oxidation in the albumin hydrophobic core and specific transfer of NO(+) to LMW thiols. Albumin-mediated S-nitrosylation and its vasodilatory effect directly depend on the concentration of circulating LMW thiols. Results suggest that the hydrophobic phase formed by albumin serves as a major reservoir of NO(.) and its reactive oxides and controls the dynamics of NO(.)-dependant processes in the vasculature
— id: 32238, year: 2002, vol: 99, page: 5913, stat: Journal Article,

Isolation and characterization of sigma(70)-retaining transcription elongation complexes from Escherichia coli
Bar-Nahum G; Nudler E
2001 Aug 24;106(4):443-451, Cell
sigma(70) subunit is thought to be released from the core RNA polymerase (RNAP) upon the transition from initiation to elongation or shortly afterward. Here, we identify a population of RNAP from E. coli that retains sigma(70) throughout elongation. The relative amount of this population appears to depend on cellular growth and reaches its maximum during the stationary phase. The proportion of sigma(70)-retaining elongation complexes (EC-sigma(70)) is invariant with various promoters or distances from the transcription start site. EC-sigma(70) responds to pauses, intrinsic terminators, and the elongation factor NusA similarly to EC without sigma(70). However, EC-sigma(70) has a substantially higher ability to support multiple rounds of transcription at certain promoters, suggesting its profound role in gene expression and regulation in bacteria
— id: 26677, year: 2001, vol: 106, page: 443, stat: Journal Article,

Control of intrinsic transcription termination by N and NusA: the basic mechanisms
Gusarov I; Nudler E
2001 Nov 16;107(4):437-449, Cell
Intrinsic transcription termination plays a crucial role in regulating gene expression in prokaryotes. After a short pause, the termination signal appears in RNA as a hairpin that destabilizes the elongation complex (EC). We demonstrate that negative and positive termination factors control the efficiency of termination primarily through a direct modulation of hairpin folding and, to a much lesser extent, by changing pausing at the point of termination. The mechanism controlling hairpin formation at the termination point relies on weak protein interactions with single-stranded RNA, which corresponds to the upstream portion of the hairpin. Escherichia coli NusA protein destabilizes these interactions and thus promotes hairpin folding and termination. Stabilization of these contacts by phage lambda N protein leads to antitermination
— id: 26542, year: 2001, vol: 107, page: 437, stat: Journal Article,

An autocatalytic mechanism of protein nitrosylation
Nedospasov A; Rafikov R; Beda N; Nudler E
2000 Dec 5;97(25):13543-13548, Proceedings of the National Academy of Sciences of the United States of America
Nitros(yl)ation is a widespread protein modification that occurs during many physiological and pathological processes. It can alter both the activity and function of a protein. Nitric oxide (( small middle dot)NO) has been implicated in this process, but its mechanism remained uncertain. ( small middle dot)NO is unable to react with nucleophiles under oxygen-free conditions, suggesting that its higher oxides, such as N(2)O(3), were actually nitrosylating agents. However, low concentrations and short lifespans of these species in vivo raise the question of how they could efficiently locate target proteins. Here we demonstrate that at physiological concentrations of ( small middle dot)NO, N(2)O(3) forms inside protein-hydrophobic cores and causes nitrosylation within the protein interior. This mechanism of protein modification has not been characterized, because all previously described mechanisms (e.g., phosphorylation, acetylation, ADP-ribosylation, etc.) occur via attack on a protein by an external modification agent. Oxidation of ( small middle dot)NO to N(2)O(3) is facilitated by micellar catalysis, which is mediated by the hydrophobic phase of proteins. Thus, a target protein seems to be a catalyst of its own nitrosylation. One of the applications of this finding, as we report here, is the design of specific hydrophobic compounds whose cooperation with ( small middle dot)NO and O(2) allows the rapid inactivation of target enzymes to occur
— id: 17430, year: 2000, vol: 97, page: 13543, stat: Journal Article,

The mechanism of intrinsic transcription termination
Gusarov I; Nudler E
1999 Apr;3(4):495-504, Molecular cell
In bacteria, an intrinsic transcription termination signal appears in RNA as a hairpin followed by approximately eight uridines (U stretch) at the 3' terminus. This signal leads to rapid dissociation of the ternary elongation complex (TEC) into RNA, DNA, and an RNA polymerase. We demonstrate that the hairpin inactivates and then destabilizes TEC by weakening interactions in the RNA-DNA hybrid-binding site and the RNA-binding site that hold TEC together. Formation of the hairpin is restricted to the moment when TEC reaches the point of termination and depends upon melting of four to five hybrid base pairs that follow the hairpin's stem. The U stretch-induced pausing at the point of termination is crucial, providing additional time for hairpin formation. These results explain the mechanism of termination and aid in understanding of how cellular factors modulate this process
— id: 56433, year: 1999, vol: 3, page: 495, stat: Journal Article,

Transcription elongation: structural basis and mechanisms
Nudler E
1999 Apr 23;288(1):1-12, Journal of molecular biology
A ternary complex composed of RNA polymerase (RNAP), DNA template, and RNA transcript is the central intermediate in the transcription cycle responsible for the elongation of the RNA chain. Although the basic biochemistry of RNAP functioning is well understood, little is known about the underlying structural determinants. The absence of high- resolution structural data has hampered our understanding of RNAP mechanism. However, recent work suggests a structure-function model of the ternary elongation complex, if not at a defined structural level, then at least as a conceptual view, such that key components of RNAP are defined operationally on the basis of compelling biochemical, protein chemical, and genetic data. The model has important implications for mechanisms of transcription elongation and also for initiation and termination.
— id: 6118, year: 1999, vol: 288, page: 1, stat: Journal Article,

Mechanistic model of the elongation complex of Escherichia coli RNA polymerase
Korzheva N; Mustaev A; Nudler E; Nikiforov V; Goldfarb A
1998 ;63(25):337-345, Cold Spring Harbor symposia on quantitative biology
— id: 17431, year: 1998, vol: 63, page: 337, stat: Journal Article,

Spatial organization of transcription elongation complex in Escherichia coli
Nudler E; Gusarov I; Avetissova E; Kozlov M; Goldfarb A
1998 Jul 17;281(5375):424-428, Science
During RNA synthesis in the ternary elongation complex, RNA polymerase enzyme holds nucleic acids in three contiguous sites: the double-stranded DNA-binding site (DBS) ahead of the transcription bubble, the RNA-DNA heteroduplex-binding site (HBS), and the RNA-binding site (RBS) upstream of HBS. Photochemical cross-linking allowed mapping of the DNA and RNA contacts to specific positions on the amino acid sequence. Unexpectedly, the same protein regions were found to participate in both DBS and RBS. Thus, DNA entry and RNA exit occur close together in the RNA polymerase molecule, suggesting that the three sites constitute a single unit. The results explain how RNA in the integrated unit RBS-HBS-DBS may stabilize the ternary complex, whereas a hairpin in RNA result in its dissociation
— id: 7720, year: 1998, vol: 281, page: 424, stat: Journal Article,

The RNA-DNA hybrid maintains the register of transcription by preventing backtracking of RNA polymerase
Nudler E; Mustaev A; Lukhtanov E; Goldfarb A
1997 Apr 4;89(1):33-41, Cell
An 8-9 bp RNA-DNA hybrid in the transcription elongation complex is essential for keeping the RNA 3' terminus engaged with the active site of E. coli RNA polymerase (RNAP). Destabilization of the hybrid leads to detachment of the transcript terminus, RNAP backtracking, and shifting of the hybrid upstream. Eventually, the exposed 3' segment of RNA can be removed through transcript cleavage. At certain sites, cycles of unwinding-rewinding of the hybrid are coupled to reverse-forward sliding of the transcription elongation complex. This explains apparent discontinuous elongation, which was previously interpreted as contraction and expansion of an RNAP molecule (inch-worming). Thus, the 3'-proximal RNA-DNA hybrid plays the dual role of keeping the active site in register with the template and sensing the helix-destabilizing mismatches in RNA, launching correction through backtracking and cleavage
— id: 17432, year: 1997, vol: 89, page: 33, stat: Journal Article,

Histidine-tagged RNA polymerase of Escherichia coli and transcription in solid phase
Kashlev M; Nudler E; Severinov K; Borukhov S; Komissarova N; Goldfarb A
1996 ;274(5272):326-334, Methods in enzymology
— id: 17434, year: 1996, vol: 274, page: 326, stat: Journal Article,

Transcription processivity: protein-DNA interactions holding together the elongation complex
Nudler E; Avetissova E; Markovtsov V; Goldfarb A
1996 Jul 12;273(5272):211-217, Science
The elongation of RNA chains during transcription occurs in a ternary complex containing RNA polymerase (RNAP), DNA template, and nascent RNA. It is shown here that elongating RNAP from Escherichia coli can switch DNA templates by means of end-to-end transposition without loss of the transcript. After the switch, transcription continues on the new template. With the use of defined short DNA fragments as switching templates, RNAP-DNA interactions were dissected into two spatially distinct components, each contributing to the stability of the elongating complex. The front (F) interaction occurs ahead of the growing end of RNA. This interaction is non-ionic and requires 7 to 9 base pairs of intact DNA duplex. The rear (R) interaction is ionic and requires approximately six nucleotides of the template DNA strand behind the active site and one nucleotide ahead of it. The nontemplate strand is not involved. With the use of protein-DNA crosslinking, the F interaction was mapped to the conserved zinc finger motif in the NH2-terminus of the beta' subunit and the R interaction, to the COOH-terminal catalytic domain of the beta subunit. Mutational disruption of the zinc finger selectively destroyed the F interaction and produced a salt-sensitive ternary complex with diminished processivity. A model of the ternary complex is proposed here that suggests that trilateral contacts in the active center maintain the nonprocessive complex, whereas a front-end domain including the zinc finger ensures processivity
— id: 17433, year: 1996, vol: 273, page: 211, stat: Journal Article,

Coupling between transcription termination and RNA polymerase inchworming
Nudler E; Kashlev M; Nikiforov V; Goldfarb A
1995 May 5;81(3):351-357, Cell
Advancement of RNA polymerase of E. coli occurs in alternating laps of monotonic and inchworm-like movement. Cycles of inchworming are encoded in DNA and involve straining and relaxation of the ternary complex accompanied by characteristic leaping of DNA and RNA footprints. We demonstrate that the oligo(T) tract that constitutes a normal part of transcription terminators acts as an inchworming signal so that the leap coincides with the termination event. Prevention of leaping with a roadblock of cleavage-defective EcoRI protein results in suppression of RNA chain release at a termination site. The results indicate that straining and relaxation of RNA polymerase are steps in the termination mechanism
— id: 17435, year: 1995, vol: 81, page: 351, stat: Journal Article,

Motion and enzymatic degradation of DNA in the atomic force microscope
Bezanilla M; Drake B; Nudler E; Kashlev M; Hansma PK; Hansma HG
1994 Dec;67(6):2454-2459, Biophysical journal
The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length
— id: 17436, year: 1994, vol: 67, page: 2454, stat: Journal Article,

Discontinuous mechanism of transcription elongation
Nudler E; Goldfarb A; Kashlev M
1994 Aug 5;265(5173):793-796, Science
During transcription elongation, three flexibly connected parts of RNA polymerase of Escherichia coli advance along the template so that the front-end domain is followed by the catalytic site which in turn is followed by the RNA product binding site. The advancing enzyme was found to maintain the same conformation throughout extended segments of the transcribed region. However, when the polymerase traveled across certain DNA sites that seemed to briefly anchor the front-end domain, cyclic shifting of the three parts, accompanied by buildup and relief of internal strain, was observed. Thus, elongation proceeded in alternating laps of monotonous and inchworm-like movement with the flexible RNA polymerase configuration being subject to direct sequence control
— id: 17437, year: 1994, vol: 265, page: 793, stat: Journal Article,

Bacteriophage T4 Alc protein: a transcription termination factor sensing local modification of DNA
Kashlev M; Nudler E; Goldfarb A; White T; Kutter E
1993 Oct 8;75(1):147-154, Cell
Bacteriophage T4 Alc protein participates in shutting off host transcription after infection of E. coli. It is demonstrated that Alc acts as a site-specific termination factor. The Alc sites occur frequently in E. coli DNA, resulting in early cessation of elongation in several tested transcription units. Alc-dependent termination requires unimpeded propagation of the elongating complex as it approaches the Alc site. Temporary halting of RNA polymerase within 10-15 bp before the Alc site prevents termination. Bacteriophage T4 transcription is protected from the action of Alc by overall substitution of cytosine with 5-hydroxymethyl cytosine in T4 DNA. In vitro methylation of CpG sequences in the vicinity of an Alc site abolishes the effect of Alc. Thus, Alc-dependent termination involves local sensing of the state of cytosine modification and a short-term 'memory' of recent pausing
— id: 17438, year: 1993, vol: 75, page: 147, stat: Journal Article,

Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli
Gragerov A; Nudler E; Komissarova N; Gaitanaris GA; Gottesman ME; Nikiforov V
1992 Nov 1;89(21):10341-10344, Proceedings of the National Academy of Sciences of the United States of America
Newly synthesized proteins aggregate extensively in Escherichia coli rpoH mutants, which are deficient in the heat shock proteins (hsp). Overproduction of either GroEL and GroES or DnaK and DnaJ prevents aggregation. If expressed together, the four hsp are effective at physiological concentrations. Our data suggest that the GroEL and GroES proteins and the DnaK and DnaJ proteins have complementary functions in the folding and assembly of most proteins
— id: 17439, year: 1992, vol: 89, page: 10341, stat: Journal Article,