Translocation mechanism of the multisubunit RNA polymerase

The expression and replication of genetic information forms the basis of life. The first step of gene expression, the transcription of DNA into RNA, is carried out in all organisms by multisubunit RNA polymerase enzymes (RNAP). During transcription, RNAP maintains 10 nucleotides of unwound DNA, the “transcription bubble”, allowing the use of one DNA strand as a template for RNA synthesis. The enzymatic machinery of RNAP couples the synthesis of RNA from nucleoside triphosphate substrates to the unwinding of the double helical DNA and the movement (translocation) along the DNA. After each transcribed nucleotide RNAP first occupies the “pre-translocated state”, after which RNAP and the transcription bubble thermally fluctuate either in the forward or backward direction. Upon forward movement, RNAP occupies the “post-translocated state” where it can bind the next nucleoside triphosphate substrate and catalyze the synthesis of RNA. Backward movement, in turn, forms the “backtracked state”, which blocks the active site in the enzyme and pauses the RNA synthesis. The molecular transitions during the translocation motion also serve as targets for regulating gene expression by protein factors and signals in the transcribed DNA. The RNAP translocation states have been characterized both structurally and biochemically, yet, the conformational dynamics of the RNAP and nucleic acids that control the translocation of RNAP still remain poorly understood. This gap in understanding is partially due to a lack of methods to directly monitor translocation with sufficient spatial and temporal resolution. Consequently, the detailed principles of the regulation of transcript synthesis remain to be discovered. Furthermore, a number of natural and synthetic antibiotics inhibit transcription by interfering with the movement of RNAP along the DNA. Thus, understanding the mechanisms of RNA synthesis and RNAP translocation as well as how they are inhibited by existing antibiotics provide routes and strategies for developing new antimicrobial drugs. In this thesis, I present the development of biophysical methods to measure Escherichia coli RNAP translocation in a time scale of milliseconds and with a resolution of one base pair (~3.4 Å). The methods rely on labelling the transcribed DNA with fluorescent nucleotide base analogues. When incorporated into suitable positions in the transcription bubble, the environment of the fluorophores changes with RNAP translocation, which can be detected as a change in the fluorescence intensity. We dissected the catalytic mechanism of RNAP by performing the fluorescent translocation measurements in parallel with assays that monitored the binding of the nucleoside triphosphate substrate into the active site of RNAP, the incorporation of the nucleotide into the RNA and the liberation of the by-product pyrophosphate (PPi). With this approach we were able to kinetically separate, for the first time, the individual steps of the RNAP catalytic cycle and established that RNAP spends a 30-80% share of the total time of the catalytic cycle in the translocation step. We employed the methods described above to study the thermodynamic and conformational basis of RNAP translocation. A key finding was that translocation is controlled by the opening and closing of the RNAP active site. The closing of the active site stabilized the pre-translocated state, whereas the opening of the active site facilitated forward translocation to the intrinsically more stable post-translocated state. PPi binding and release modulated translocation by favoring active site closing and opening, respectively. These findings were corroborated by the identification of RNAP variants with altered translocation and/or PPi release properties. Moreover, these investigations were inseparably connected to the identification of the inhibitory mechanisms of small molecules. We found that the RNAP inhibitor tagetitoxin prevented forward translocation by stabilizing the closed active site, whereas CBR703 destabilized the closed active site facilitating forward translocation, which, however, made RNAP hypersensitive to DNA encoded regulatory signals. We further dissected the movements of the DNA and the RNA during translocation with a combination of kinetic assays and the structural analysis of the transcription bubble using nucleic acid photo-crosslinking techniques. Our observations indicated that translocation is a multi-step process. Nucleotide addition by RNAP was followed by the opening of the active site and the translocation of the RNA, however, the DNA translocated only partially at this stage, suggesting the presence of an intermediate translocation state between the canonical pre- and post-translocation states. The RNAP catalytic cycle was then completed by the translocation of the DNA. Interestingly, the transcribed sequence could modulate the rate of DNA translocation to an extent that it became the rate-limiting step of the RNAP catalytic cycle. We provide experimental evidence for the two-step translocation mechanism and demonstrate how it can explain the control of RNA synthesis by the DNA sequence. Finally, we studied the mechanism and regulation of RNAP backtracking by measuring the kinetics of entry into the backtracked state by Gre-factor mediated RNA cleavage. RNAP backtracking was found to be facilitated by the melting of the DNA at the rear end of the transcription bubble. The universally conserved transcription factor NusG inhibited RNAP backtracking by stabilizing the DNA base pairs at the rear end of the transcription bubble, explaining at the molecular level, how NusG speeds up the rate of RNA synthesis. In another study, we investigated the structural basis of backtracking by incorporating a base analogue fluorophore into the RNA. Upon entry into the backtracked state the end of the RNA separated from the DNA giving rise to a fluorescence signal. Unexpectedly, the closing of the active site stabilized the backtracked RNA and prolonged the life-time of the backtracked pause. We found that the backtracked RNA binds in a pocket near the RNAP active site (the so-called E-site) that overlaps with the PPi and tagetitoxin binding sites. Together these interactions inhibited the forward translocation of RNAP by a factor of ~100. Although the prolonged occupancy of the RNA in the backtracked state facilitated transcriptional proofreading, the closed active site interfered with the RNA cleavage by the Gre-factors. Thus, our observations indicate that the binding of the backtracked RNA in the E-site may contribute to gene regulation by modulating the rate of RNA synthesis. The results present advances in the quantitative description of the catalytic mechanism of RNAP. Our observations reveal important features of RNAP operation as a molecular machine controlled by nucleic acids, transcription factors and small molecules, and open new mechanistic questions to be pursued. The methods presented here are applicable to similar studies in Archaeal and Eukaryotic transcription machineries.TIIVISTELMÄ Elämä perustuu geneettisen perimän ilmentymiseen ja kopioitumiseen. Kaikissa eliöissä geenien ilmenemisen ensimmäinen vaihe on DNA:n kopioituminen RNA:ksi eli transkriptio. Transkriptiota katalysoivat RNA-polymeraasientsyymit (RNAP). Transkription aikana RNAP erottaa kaksinauhaisen DNA:n juosteet toisistaan ylläpitämällä 10 nukleotidiä sisältävää ”transkriptiokuplaa”, jolloin toista DNA-juostetta voidaan käyttää RNA:n synteesin ohjeena. RNAP:n entsyymikoneisto kytkee yhteen RNA-synteesin nukleosiditrifosfaatti-substraateista, DNA:n kaksoiskierteen avaamisen ja liikkeen (translokaation) DNA:ta pitkin. Jokaisen nukleotidin liittämisen jälkeen RNAP on ”pre-translokaatiotilassa”, josta se lämpöliikkeen vaikutuksesta siirtyy joko eteen- tai taaksepäin. Eteenpäin suuntautuvan liikkeen jälkeen RNAP päätyy ”post-translokaatiotilaan”, jossa se voi sitoa seuraavan substraatin ja katalysoida RNA:n synteesiä. Taaksepäin suuntautuva liike puolestaan synnyttää ”peruutustilan”, jossa RNA:n synteesi estyy. RNAP:iin sitoutuvat toiset proteiinit ja DNA:n signaalit voivat ohjata näitä liikkeitä ja siten säädellä geenien ilmenemistä. Vaikka RNAP on perusteellisesti kuvattu sekä rakenteellisesti että biokemiallisesti translokaation eri vaiheissa, näiden tilojen välinen dynamiikka sekä niitä välittävät konformaatiomuutokset ovat edelleen huonosti tunnettuja. Joukko synteettisiä ja luonnosta eristettyjä antibiootteja ehkäisee mikrobien kasvua estämällä RNAP:n liikkumista DNA:ta pitkin. Uusien antibioottien kehittämiseksi on tärkeää ymmärtää RNA-synteesin ja translokaation mekanismeja sekä selvittää, miten tunnetut antibiootit estävät RNAP:a katalysoimasta yllä mainittuja reaktiovaiheita. Transkription säätelyyn liittyvien molekyylitason periaatteiden selvittämistä on hidastanut olennaisesti puute menetelmistä, joilla translokaatiota voidaan suoraan havainnoida. Väitöskirjassani kehitin biofysikaalisia menetelmiä, joilla voitiin mitata Escherichia coli -bakteerista eristetyn RNAP:n translokaatiota millisekuntien aikaskaalassa yhden emäsparin (~3.4 Å) tarkkuudella. Menetelmät perustuvat fluoresoivien emäsanalogien sijoittamiseen DNA:han. Sopivissa kohdissa transkriptiokuplaa fluoroforien ympäristö muuttuu translokaation seurauksena, ja tämä voidaan havaita fluoresenssisignaalin kasvuna. Tutkimme RNAP:n katalyysimekanismia mittaamalla translokaation lisäksi nukleotidin sitoutumista aktiiviseen keskukseen, nukleotidin liittymistä RNA:han ja pyrofosfaatin (PPi) irtoamista. Näin pystyimme ensimmäisinä erottamaan toisistaan RNAP:n katalyysisyklin erilliset reaktiovaiheet ja mittaamaan translokaation kestoksi 30-80% katalyysisyklin kokonaisajasta. Menetelmien avulla tutkimme translokaation termodynaamista perustaa. Keskeinen havaintomme oli, että translokaatiota säätelee RNAP:n aktiivisen keskuksen avautuminen ja sulkeutuminen. Aktiivisen keskuksen sulkeutuminen stabiloi pre-translokaatiotilaa, kun taas sen avautuminen mahdollisti eteenpäin suuntautuvan liikkeen muutoin stabiilimpaan post-translokaatiotilaan. PPi:n sitoutuminen ja irtoaminen kytkeytyivät translokaatioon, edellinen suosimalla aktiivisen keskuksen sulkeutumista ja jälkimmäinen sen avautumista. Havaintojemme vahvistukseksi tunnistimme RNAP:sta rakenteellisia mutaatioita, jotka vaikuttivat translokaatioon ja PPi:n irtoamiseen. Nämä tutkimukset liittyivät myös selvitykseen mekanismeista, joilla pienmolekyylit estävät RNAP:n katalyysiä. Havaitsimme, että tagetitoksiini esti translokaatiota stabiloimalla suljettua aktiivista keskusta, kun taas CBR703 vaikutti päinvastaisesti ja edisti translokaatiota. Jälkimmäisessä tapauksessa RNAP kuitenkin muuttui samalla yliherkäksi DNA:n transkriptiota sääteleville signaaleille, mikä hidasti RNA:n synteesinopeutta. Selvitimme translokaation aikana tapahtuvia DNA:n ja RNA:n liikkeitä kineettisten mittausten lisäksi biokemiallisilla menetelmillä, joilla voitiin analysoida transkriptiokuplan rakennetta. Tulosten mukaan translokaatioon sisältyy useita vaiheita. Nukleotidin liittymistä seurasi aktiivisen keskuksen avautuminen ja RNA:n translokaatio, mutta DNA ei kuitenkaan translokoitunut tässä vaiheessa vielä kokonaan viitaten siihen, että tyypillisten pre- ja post-translokaatiotilojen lisäksi on olemassa niiden välivaihe. Tämän jälkeen seurasi katalyysisyklin päättävä DNA:n täydellinen translokaatio. Mielenkiintoista oli se, että DNA:n emäsjärjestys vaikutti DNA:n translokaationopeuteen jopa toisinaan siinä määrin, että siitä tuli koko katalyysisyklin nopeutta rajoittava vaihe. Kokeelliset tuloksemme tukevat kaksivaiheista translokaatiomallia ja osoittavat, miten malli voi selittää DNA:n emäsjärjestyksen erilaiset vaikutukset RNA-synteesiin. Tutkimme myös taaksepäin suuntautuvaa translokaatiota ja sen säätelyä mittamalla RNAP:n peruutustilan muodostumista. Tässä hyödynsimme Gre-proteiineja, jotka edistävät RNA:n leikkautumista RNAP:n peruutustilassa. DNA:n kaksoiskierteen avautuminen transkriptiokuplan perässä edisti taaksepäin suuntautuvaa translokaatiota. Evolutiivisesti hyvin konservoitunut säätelyproteiini NusG vähensi peruutustilaan joutumista stabiloimalla DNA:n emäspareja transkriptiokuplan perässä. Tämä tulos selittää molekyylitasolla, mihin perustuu NusG:n kyky nopeuttaa RNA:n synteesiä. Toisessa osatyössä tutkimme RNAP:n peruutustilan rakennetta sijoittamalla fluoresoivan emäsanalogin RNA:n päähän. Fluoresenssi kasvoi, kun RNA:n pää erkani DNA:sta peruutustilan muodostuessa. Yllättäen aktiivisen keskuksen sulkeutuminen stabiloi peruutustilaa ja näin pitkitti RNA-synteesin pysähdystä. Havaitsimme, että peruutustilaan joutunut RNA sitoutui aktiivisen keskuksen lähellä sijaitsevaan taskuun, joka sivuaa PPi:n ja tagetitoksiinin sitoutumiskohtia. Yhdessä nämä vuorovaikutukset hidastivat RNAP:n eteenpäin suuntautuvaa translokaationopeutta noin sadasosaan alkuperäisestä. Vaikka RNA:n pitkittynyt sitoutuminen peruutustilaan edisti RNA:n oikolukua, sulkeutunut aktiivinen keskus esti Gre-välitteistä RNA:n leikkautumista. Kuvaamamme peruutustilan vuorovaikutukset voivat täten ensisijaisesti vaikuttaa RNA-synteesin nopeuteen, ja edelleen geenien ilmenemisen säätelyyn. Väitöskirjani tulokset edistävät kvantitatiivista ymmärrystä RNAP:n katalyysimekanismeista. Havaintomme valaisevat RNAP:n molekyylikoneiston toimintaa ja periaatteita, joilla nukleiinihappo-rakenteet, transkriptiota säätelevät proteiinit ja pienmolekyylit vaikuttavat RNAP:n aktiivisuuteen. Esitetyt tutkimusmenetelmät ovat sovellettavissa myös arkkibakteerien ja eukaryoottien transkriptiokoneistojen tutkimiseen

NusG inhibits RNA polymerase backtracking by stabilizing the minimal transcription bubble

Universally conserved factors from NusG family bind at the upstream fork junction of transcription elongation complexes and modulate RNA synthesis in response to translation, processing, and folding of the nascent RNA. Escherichia coli NusG enhances transcription elongation in vitro by a poorly understood mechanism. Here we report that E. coli NusG slows Gre factor-stimulated cleavage of the nascent RNA, but does not measurably change the rates of single nucleotide addition and translocation by a non-paused RNA polymerase. We demonstrate that NusG slows RNA cleavage by inhibiting backtracking. This activity is abolished by mismatches in the upstream DNA and is independent of the gate and rudder loops, but is partially dependent on the lid loop. Our comprehensive mapping of the upstream fork junction by base analogue fluorescence and nucleic acids crosslinking suggests that NusG inhibits backtracking by stabilizing the minimal transcription bubble.</p

Dynamic phosphorus and nitrogen yield response model for economic optimisation

This paper provides an approach for modelling joint impact of two main nutrients in crop production for situations where there are available separate datasets for nitrogen and phosphorus fertiliser field experiments. Developing yield response models for Finnish spring barley crops (Hordeum vulgare L.) for clay and coarse soils and applying the models for dynamic economic analysis demonstrate the modelling approach. Model selection is based on iterative elimination from a wide diversity of plausible model formulations. Nonlinear weighted least squares method was utilised in estimation of the yield response models and dynamic programming was utilised in economic analysis. Our results suggest that fertiliser recommendations can be insufficient if soil phosphorus dynamics are ignored. Further, the optimal fertilisation rates for nitrogen and phosphorus, as well as the economic alternative costs of agri-environmental programmes depend on the soil texture of production area. Therefore, the efficiency of such programmes could be improved by targeting different fertilisation limits for different soil textures. In addition, uncertainty analysis revealed that the parameter uncertainty had a greater effect on the model output than the structural uncertainty. Further, the interaction of nitrogen and phosphorus fertilisers appeared to be a factor of relatively minor importance. The modelling approach and the model structure can be extended to other geographical areas, given that adequate datasets are available.This paper provides an approach for modelling joint impact of two main nutrients in crop production for situations where there are available separate datasets for nitrogen and phosphorus fertiliser field experiments. Developing yield response models for Finnish spring barley crops (Hordeum vulgare L.) for clay and coarse soils and applying the models for dynamic economic analysis demonstrate the modelling approach. Model selection is based on iterative elimination from a wide diversity of plausible model formulations. Nonlinear weighted least squares method was utilised in estimation of the yield response models and dynamic programming was utilised in economic analysis. Our results suggest that fertiliser recommendations can be insufficient if soil phosphorus dynamics are ignored. Further, the optimal fertilisation rates for nitrogen and phosphorus, as well as the economic alternative costs of agri-environmental programmes depend on the soil texture of production area. Therefore, the efficiency of such programmes could be improved by targeting different fertilisation limits for different soil textures. In addition, uncertainty analysis revealed that the parameter uncertainty had a greater effect on the model output than the structural uncertainty. Further, the interaction of nitrogen and phosphorus fertilisers appeared to be a factor of relatively minor importance. The modelling approach and the model structure can be extended to other geographical areas, given that adequate datasets are available

Phosphorus and Nitrogen Yield Response Models for Dynamic Bio-Economic Optimization: An Empirical Approach

Nitrogen (N) and phosphorus (P) are both essential plant nutrients. However, their joint response to plant growth is seldom described by models. This study provides an approach for modeling the joint impact of inorganic N and P fertilization on crop production, considering the P supplied by the soil, which was approximated using the soil test P (STP). We developed yield response models for Finnish spring barley crops (Hordeum vulgare L.) for clay and coarse-textured soils by using existing extensive experimental datasets and nonlinear estimation techniques. Model selection was based on iterative elimination from a wide diversity of plausible model formulations. The Cobb-Douglas type model specification, consisting of multiplicative elements, performed well against independent validation data, suggesting that the key relationships that determine crop responses are captured by the models. The estimated models were extended to dynamic economic optimization of fertilization inputs. According to the results, a fair STP level should be maintained on both coarse-textured soils (9.9 mg L-1 a(-1)) and clay soils (3.9 mg L-1 a(-1)). For coarse soils, a higher steady-state P fertilization rate is required (21.7 kg ha(-1) a(-1)) compared with clay soils (6.75 kg ha(-1) a(-1)). The steady-state N fertilization rate was slightly higher for clay soils (102.4 kg ha(-1) a(-1)) than for coarse soils (95.8 kg ha(-1) a(-1)). This study shows that the iterative elimination of plausible functional forms is a suitable method for reducing the effects of structural uncertainty on model output and optimal fertilization decisions.Peer reviewe

Birth of a poly(A) tail: mechanisms and control of mRNA polyadenylation

During their synthesis in the cell nucleus, most eukaryotic mRNAs undergo a two-step 3′-end processing reaction in which the pre-mRNA is cleaved and released from the transcribing RNA polymerase II and a polyadenosine (poly(A)) tail is added to the newly formed 3′-end. These biochemical reactions might appear simple at first sight (endonucleolytic RNA cleavage and synthesis of a homopolymeric tail), but their catalysis requires a multi-faceted enzymatic machinery, the cleavage and polyadenylation complex (CPAC), which is composed of more than 20 individual protein subunits. The activity of CPAC is further orchestrated by Poly(A) Binding Proteins (PABPs), which decorate the poly(A) tail during its synthesis and guide the mRNA through subsequent gene expression steps. Here, we review the structure, molecular mechanism, and regulation of eukaryotic mRNA 3′-end processing machineries with a focus on the polyadenylation step. We concentrate on the CPAC and PABPs from mammals and the budding yeast, Saccharomyces cerevisiae, because these systems are the best-characterized at present. Comparison of their functions provides valuable insights into the principles of mRNA 3′-end processing. </p

Role of the trigger loop in translesion RNA synthesis by bacterial RNA polymerase

DNA lesions can severely compromise transcription and block RNA synthesis by RNA polymerase (RNAP), leading to subsequent recruitment of DNA repair factors to the stalled transcription complex. Recent structural studies have uncovered molecular interactions of several DNA lesions within the transcription elongation complex. However, little is known about the role of key elements of the RNAP active site in translesion transcription. Here, using recombinantly expressed proteins,in vitrotranscription, kinetic analyses, andin vivocell viability assays, we report that point amino acid substitutions in the trigger loop, a flexible element of the active site involved in nucleotide addition, can stimulate translesion RNA synthesis byEscherichia coliRNAP without altering the fidelity of nucleotide incorporation. We show that these substitutions also decrease transcriptional pausing and strongly affect the nucleotide addition cycle of RNAP by increasing the rate of nucleotide addition but also decreasing the rate of translocation. The secondary channel factors DksA and GreA modulated translesion transcription by RNAP, depending on changes in the trigger loop structure. We observed that although the mutant RNAPs stimulate translesion synthesis, their expression is toxicin vivo, especially under stress conditions. We conclude that the efficiency of translesion transcription can be significantly modulated by mutations affecting the conformational dynamics of the active site of RNAP, with potential effects on cellular stress responses and survival

Loimijoki-projektin raportti 1991-1997 : ympäristöhankkeen eteneminen Loimijokilaakson maatiloilla ja jokirannoilla

vokMK

GreA and GreB Enhance Expression of Escherichia coil RNA Polymerase Promoters in a Reconstituted Transcription-Translation System

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions, making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology

Three-layered control of mRNA poly(A) tail synthesis in Saccharomyces cerevisiae

Biogenesis of most eukaryotic mRNAs involves the addition of an untemplated polyadenosine (pA) tail by the cleavage and polyadenylation machinery. The pA tail, and its exact length, impacts mRNA stability, nuclear export, and translation. To define how polyadenylation is controlled in S. cerevisiae, we have used an in vivo assay capable of assessing nuclear pA tail synthesis, analyzed tail length distributions by direct RNA sequencing, and reconstituted polyadenylation reactions with purified components. This revealed three control mechanisms for pA tail length. First, we found that the pA binding protein (PABP) Nab2p is the primary regulator of pA tail length. Second, when Nab2p is limiting, the nuclear pool of Pab1p, the second major PABP in yeast, controls the process. Third, when both PABPs are absent, the cleavage and polyadenylation factor (CPF) limits pA tail synthesis. Thus, Pab1p and CPF provide fail-safe mechanisms to a primary Nab2p-dependent pathway, thereby preventing uncontrolled polyadenylation and allowing mRNA export and translation