Characterization of RNA sequence determinants and antideterminants of processing reactivity for a minimal substrate of Escherichia coli ribonuclease III

Members of the ribonuclease III family are the primary agents of double-stranded (ds) RNA processing in prokaryotic and eukaryotic cells. Bacterial RNase III orthologs cleave their substrates in a highly site-specific manner, which is necessary for optimal RNA function or proper decay rates. The processing reactivities of Escherichia coli RNase III substrates are determined in part by the sequence content of two discrete double-helical elements, termed the distal box (db) and proximal box (pb). A minimal substrate of E.coli RNase III, μR1.1 RNA, was characterized and used to define the db and pb sequence requirements for reactivity and their involvement in cleavage site selection. The reactivities of μR1.1 RNA sequence variants were examined in assays of cleavage and binding in vitro. The ability of all examined substitutions in the db to inhibit cleavage by weakening RNase III binding indicates that the db is a positive determinant of RNase III recognition, with the canonical UA/UG sequence conferring optimal recognition. A similar analysis showed that the pb also functions as a positive recognition determinant. It also was shown that the ability of the GC or CG bp substitution at a specific position in the pb to inhibit RNase III binding is due to the purine 2-amino group, which acts as a minor groove recognition antideterminant. In contrast, a GC or CG bp at the pb position adjacent to the scissile bond can suppress cleavage without inhibiting binding, and thus act as a catalytic antideterminant. It is shown that a single pb+db ‘set’ is sufficient to specify a cleavage site, supporting the primary function of the two boxes as positive recognition determinants. The base pair sequence control of reactivity is discussed within the context of new structural information on a post-catalytic complex of a bacterial RNase III bound to the cleaved minimal substrate

Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Escherichia coli ribonuclease III (RNase III; EC 3.1.24) is a double-stranded(ds)-RNA-specific endonuclease with key roles in diverse RNA maturation and decay pathways. E.coli RNase III is a member of a structurally distinct superfamily that includes Dicer, a central enzyme in the mechanism of RNA interference. E.coli RNase III requires a divalent metal ion for activity, with Mg(2+) as the preferred species. However, neither the function(s) nor the number of metal ions involved in catalysis is known. To gain information on metal ion involvement in catalysis, the rate of cleavage of the model substrate R1.1 RNA was determined as a function of Mg(2+) concentration. Single-turnover conditions were applied, wherein phosphodiester cleavage was the rate-limiting event. The measured Hill coefficient (n(H)) is 2.0 ± 0.1, indicative of the involvement of two Mg(2+) ions in phosphodiester hydrolysis. It is also shown that 2-hydroxy-4H-isoquinoline-1,3-dione—an inhibitor of ribonucleases that employ two divalent metal ions in their catalytic sites—inhibits E.coli RNase III cleavage of R1.1 RNA. The IC(50) for the compound is 14 μM for the Mg(2+)-supported reaction, and 8 μM for the Mn(2+)-supported reaction. The compound exhibits noncompetitive inhibitory kinetics, indicating that it does not perturb substrate binding. Neither the O-methylated version of the compound nor the unsubstituted imide inhibit substrate cleavage, which is consistent with a specific interaction of the N-hydroxyimide with two closely positioned divalent metal ions. A preliminary model is presented for functional roles of two divalent metal ions in the RNase III catalytic mechanism

Koinon: the Coexistence of Geniuses or Flight of Ideas in the Air?

Received: 4.10.2020. Accepted: 19.10.2020.Рукопись поступила в редакцию: 4.10.2020. Принята к публикации: 19.10.2020.The article by A. V. Pertsev, professor of the Ural Federal University, on the example of Weimar’s life as a “German cultural capital” at the time of I. V. Goethe (1749–1832) discusses the specifics of the organization of informal intellectual communities that defined the existing forms of culture in Europe and Russia. The paper analyzes such a humanitarian koinon as a form of coexistence and cooperation of creative people, which emerged in a particular historical situation and is supported by “enlightened authorities”. The figure of the bearer of power as a “cultural manager” and his formation of “cultural centres” following his ideas of a worthy human life are as well under consideration. As the author of the article demonstrates, the state cultural policy is concealed in such a way that its central figure is the “enlightener on the throne” himself and the “leading personnel” he has chosen. This aristocratic cultural policy is opposed to a market-democratic one with a focus on ‘mass demand’.В статье на примере жизни Веймара как «немецкой культурной столицы» времен И. В. Гёте (1749–1832) рассматривается специфика организации неформальных интеллектуальных сообществ, которые определяли ныне существующие формы культуры в Европе и в России. Такой гуманитарный koinon анализируется как возникшая в определенной исторической ситуации форма сосуществования и сотрудничества творческих людей, поддержку творчества которой обеспечивают «просвещенные власти». Рассматривается фигура носителя власти как «менеджера культуры» и формирование им «центров культуры» в соответствии с собственными представлениями о достойной человеческой жизни. Государственная культурная политика, как демонстрирует автор статьи, скроена так, что ее центральной фигурой начинает выступать сам «просветитель на троне» и подобранные им «руководящие кадры». Такая аристократическая культурная политика противостоит рыночно-демократической, которая ориентирована на «массовый спрос»

Ca2+-Sensor Neurocalcin δ and Hormone ANF Modulate ANF-RGC Activity by Diverse Pathways: Role of the Signaling Helix Domain

Prototype member of the membrane guanylate cyclase family, ANF-RGC (Atrial Natriuretic Factor Receptor Guanylate Cyclase), is the physiological signal transducer of two most hypotensive hormones ANF and BNP, and of the intracellular free Ca2+. Both the hormonal and the Ca2+-modulated signals operate through a common second messenger, cyclic GMP; yet, their operational modes are divergent. The hormonal pathways originate at the extracellular domain of the guanylate cyclase; and through a cascade of structural changes in its successive domains activate the C-terminal catalytic domain (CCD). In contrast, the Ca2+ signal operating via its sensor, myristoylated neurocalcin δ both originates and is translated directly at the CCD. Through a detailed sequential deletion and expression analyses, the present study examines the role of the signaling helix domain (SHD) in these two transduction pathways. SHD is a conserved 35-amino acid helical region of the guanylate cyclase, composed of five heptads, each meant to tune and transmit the hormonal signals to the CCD for their translation and generation of cyclic GMP. Its structure is homo-dimeric and the molecular docking analyses point out to the possibility of antiparallel arrangement of the helices. Contrary to the hormonal signaling, SHD has no role in regulation of the Ca2+- modulated pathway. The findings establish and define in molecular terms the presence of two distinct non-overlapping transduction modes of ANF-RGC, and for the first time demonstrate how differently they operate, and, yet generate cyclic GMP utilizing common CCD machinery

Membrane Guanylate Cyclase catalytic Subdomain: Structure and Linkage with Calcium Sensors and Bicarbonate

Membrane guanylate cyclase (MGC) is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD) module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC’s CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1) The CCD is a conserved 145-residue structural unit, represented by the segment V820-P964. (2) It exists as a homo-dimer and contains seven conserved catalytic elements (CEs) wedged into seven conserved motifs. (3) It also contains a conserved 21-residue neurocalcin δ-modulated structural domain, V836-L857. (4) Site-directed mutagenesis documents that each of the seven CEs governs the cyclase’s catalytic activity. (5) In contrast to the soluble and the bacterium MGC which use Mn2+-GTP substrate for catalysis, MGC CCD uses the natural Mg2+-GTP substrate. (6) Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD) to exhibit its major (∼92%) catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7) the seven CEs control each of four phototransduction pathways- -two Ca2+-sensor GCAPs-, one Ca2+-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the physiological activity of other members of MGC family

Koinon: Being-in-common, Heterology, Post-fundamentalism

Received: 27.09.2020. Accepted: 12.10.2020.Рукопись поступила в редакцию: 27.09.2020. Принята к публикации: 12.10.2020.The problem of being-in-common remains a fundamental problem of philosophy, social sciences and humanities. The purpose of this article is to analyze the changes in the nature of this problem, the incentives and prospects for its formulation and solution in the late XX — early XXI centuries. Being-incommon is increasingly shifting to the focus of human existence, acquires a decisive existential significance, acts as public opinion, and as a political motivation, and as a paradigm for the social sciences and humanities. The article argues that full awareness of the role of philosophy in describing and explaining the forms of being-in-common presupposes the need for a radical reorientation of both traditional philosophical constructions and the methodology of social and humanitarian cognition. Three dimensions of this reorientation are highlighted. Firstly, this is the “new sociality”, the undecidability between “globalization” and “mondialization”, as well as the need to deconstruct the very possibility of accepting a global or phenomenal world. Secondly, it is ontoheterology, which outlines the ways of overcoming ontological fundamentalism, building an “ontology after ontotheology”. It exposes the understanding of being-in-common, the original sociality as groundless, anarchic, which arises as a direct correlate of the experience of groundlessness of existence, unrooted in any substance or common essence. Thirdly, this is postfundamentalism as a particular paradigm, style of thinking, postulating the transformation of the practices and techniques of thinking in response to the need to reposition the multiple real as immanent in thought itself. The article concludes that the three-dimensional “space” of comprehending being-in-common sets a certain direction of philosophical and socio-humanitarian research, hidden behind them general methodological tendencies, and the relationship of these tendencies with the nature of social practice.Проблема совместного существования остается фундаментальной проблемой философии и социально-гуманитарных наук. Целью данной статьи является анализ изменений характера этой проблемы, стимулов и перспектив ее постановки и решения в конце ХХ — начале ХХI в. Совместность всё более сдвигается в средоточие человеческого бытия, приобретает решающее экзистенциальное значение, выступает и как общественное мнение, и как политическая мотивация, и как парадигма для социально-гуманитарных наук. В статье утверждается, что полное осознание роли философии в описании и объяснении форм совместного существования предполагает необходимость радикальной переориентации как традиционных философских построений, так и методологии социально-гуманитарного познания. Выделены три измерения этой переориентации. Во-первых, это «новая социальность», неразрешимость между «глобализацией» и «мондиализацией», а также необходимость деконструкции самой возможности принятия глобального или феноменального мира. Во-вторых, это онтогетерология, намечающая пути преодоления онтологического фундаментализма, построения «онтологии после онтотеологии» и обнажающая понимание совместности, исходной социальности как безосновной, анархичной, которое возникает как непосредственный коррелят опыта безосновности существования, не укорененного в какой-либо субстанции или общей сущности. В-третьих, это постфундаментализм как определенная парадигма, стилистика мышления, постулирующая трансформацию практик и техник мышления в ответ на необходимость репозиционирования множественного реального как имманентного самой мысли. В статье делается вывод о том, что трехмерное «пространство» осмысления совместности существования задает определенную направленность философских и социально-гуманитарных исследований, скрытые за ними общеметодологические тенденции, взаимосвязи этих тенденций с характером социальной практики

Regulation of gene expression in restriction-modification system Eco29kI

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR–eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes

Characterization of Aquifex aeolicus ribonuclease III and the reactivity epitopes of its pre-ribosomal RNA substrates

Ribonuclease III cleaves double-stranded (ds) structures in bacterial RNAs and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, the biochemical properties of A. aeolicus (Aa)-RNase III and the reactivity epitopes of its substrates are not known. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of ∼70–85°C, with either Mg2+ or Mn2+ supporting efficient catalysis. Small hairpins based on the stem structures associated with the Aquifex 16S and 23S rRNA precursors are cleaved at sites that are consistent with production of the immediate precursors to the mature rRNAs. Substrate reactivity is independent of the distal box sequence, but is strongly dependent on the proximal box sequence. Structural studies have shown that a conserved glutamine (Q157) in the Aa-RNase III dsRNA-binding domain (dsRBD) directly interacts with a proximal box base pair. Aa-RNase III cleavage of the pre-16S substrate is blocked by the Q157A mutation, which reflects a loss of substrate binding affinity. Thus, a highly conserved dsRBD-substrate interaction plays an important role in substrate recognition by bacterial RNase III

Bacteriophage T4 endonuclease II, a promiscuous GIY-YIG nuclease, binds as a tetramer to two DNA substrates

The oligomerization state and mode of binding to DNA of the GIY-YIG endonuclease II (EndoII) from bacteriophage T4 was studied using gel filtration and electrophoretic mobility shift assays with a set of mutants previously found to have altered enzyme activity. At low enzyme/DNA ratios all mutants except one bound to DNA only as tetramers to two DNA substrates. The putatively catalytic E118 residue actually interfered with DNA binding (possibly due to steric hindrance or repulsion between the glutamate side chain and DNA), as shown by the ability of E118A to bind stably also as monomer or dimer to a single substrate. The tetrameric structure of EndoII in the DNA–protein complex is surprising considering the asymmetry of the recognized sequence and the predominantly single-stranded nicking. Combining the results obtained here with those from our previous in vivo studies and the recently obtained crystal structure of EndoII E118A, we suggest a model where EndoII translocates DNA between two adjacent binding sites and either nicks one strand of one or both substrates bound by the tetramer, or nicks both strands of one substrate. Thus, only one or two of the four active sites in the tetramer is catalytically active at any time

Fused eco29kIR- and M genes coding for a fully functional hybrid polypeptide as a model of molecular evolution of restriction-modification systems

<p>Abstract</p> <p>Background</p> <p>The discovery of restriction endonucleases and modification DNA methyltransferases, key instruments of genetic engineering, opened a new era of molecular biology through development of the recombinant DNA technology. Today, the number of potential proteins assigned to type II restriction enzymes alone is beyond 6000, which probably reflects the high diversity of evolutionary pathways. Here we present experimental evidence that a new type IIC restriction and modification enzymes carrying both activities in a single polypeptide could result from fusion of the appropriate genes from preexisting bipartite restriction-modification systems.</p> <p>Results</p> <p>Fusion of <it>eco29kIR </it>and <it>M </it>ORFs gave a novel gene encoding for a fully functional hybrid polypeptide that carried both restriction endonuclease and DNA methyltransferase activities. It has been placed into a subclass of type II restriction and modification enzymes - type IIC. Its MTase activity, 80% that of the M.Eco29kI enzyme, remained almost unchanged, while its REase activity decreased by three times, concurrently with changed reaction optima, which presumably can be caused by increased steric hindrance in interaction with the substrate. <it>In vitro </it>the enzyme preferentially cuts DNA, with only a low level of DNA modification detected. <it>In vivo </it>new RMS can provide a 10²-fold less protection of host cells against phage invasion.</p> <p>Conclusions</p> <p>We propose a molecular mechanism of appearing of type IIC restriction-modification and M.SsoII-related enzymes, as well as other multifunctional proteins. As shown, gene fusion could play an important role in evolution of restriction-modification systems and be responsible for the enzyme subclass interconversion. Based on the proposed approach, hundreds of new type IIC enzymes can be generated using head-to-tail oriented type I, II, and III restriction and modification genes. These bifunctional polypeptides can serve a basis for enzymes with altered recognition specificities. Lastly, this study demonstrates that protein fusion may change biochemical properties of the involved enzymes, thus giving a starting point for their further evolutionary divergence.</p