Elife 7, e42166 (2018). Alteration of the absolutely conserved K239 to A in the Walker A motif resulted in the complete loss of transcription recycling and ATPase activity (Fig. Biol. 1). Overall, this structure serves as a valuable resource for structure-function studies with RNAP from the Firmicutes as well as being a reference structure to enable full understanding of the conformational changes involved in transcription complex recycling induced upon binding to HelD (below). Helicase enzymes are also critical for making a blueprint of a gene for protein production, a process called transcription. The interaction of the RNA with both the PBS and the SBS promotes ring closure and activates Rho ATPase activity and translocation in the 5 to 3 direction.8,9 As a translocase, Rho is fast (2-5 times faster than RNAP) but moderately processive, since it dissociates from the RNA after translocating 6080 nt.10,11 As a consequence, Rho-dependent termination is strongly dependent on RNAP pausing.6 Three alternative models have been proposed to explain the mechanism by which Rho induces EC dissociation once it catches up with the RNAP. 5), and even within the same organism, sequence conservation between the copies is relatively low in the SCA and CA domains (Supplementary Fig. HelD then forces the primary channel open through interaction of the CA with the clamp (Uppercut). copyright 2003-2023 Study.com. Despite considerable effort, no structure of RNAP from the low G+C Gram positives has been determined to date. Zhang, Y., Ahn, B. Y. Cell 75, 298309 e294 (2019). J. Biol. Commun. ATPase activity was determined by malachite green assay56. The end of the CA forms a relatively flat ~320 2 surface that acts as a platform to push up against the clamp, resulting in loss of contact with the DNA bound in the EC (Figs. 47, 411423 (2019). & Cramer, P. A movie of the RNA polymerase nucleotide addition cycle. Importantly, the helicase domain is sufficient for termination in vitro,24 indicating that this region retains all the properties and activities that are necessary for EC dissociation. No potential conflicts of interest were disclosed. Chem. Nucleotide substrate and polynucleotide cofactor specificities. Clemens Grimm or Utz Fischer. Elements identified as functionally, architecturally or structurally corresponding are colored according to the scheme used for the vaccinia PIC throughout this manuscript. Liu, H. & Naismith, J. H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. Cheetham, G. M. & Steitz, T. A. I would definitely recommend Study.com to my colleagues. Yang, Z. Trans. They unwind the DNA with the help of ATP hydrolysis, and thus facilitate the replication and transcription processes. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S. & Candia, O. Tan, S., Hunziker, Y., Sargent, D. F. & Richmond, T. J. Genes Dev. Frozen pellets from 8L culture were resuspended in 50ml HisA buffer (above) supplemented with EDTA-free protease inhibitor cocktail (1.8 concentration S8830, Sigma-Aldrich) at 4C, and lysed by multiple passage through an Avestin C5 homogeniser at ~25 kPa, and the lysate clarified by centrifugation at 16,000g, 4C, 20min. Its like a teacher waved a magic wand and did the work for me. For the EC, movies were collected as a series of 40 frames and a total accumulated dose of 52.2 e/2. Afonine, P. V. et al. Kostrewa, D. et al. Previous studies have also shown deletion of causes no detectable phenotype or change in gene expression profiles, and RNAP core preparations lacking have indistinguishable activity compared to those that do contain it19,20. Brueckner, F., Ortiz, J. In S. cerevisiae Sen1 forms a complex with two RNA-binding proteins, Nrd1 and Nab3, which recognize particular sequences that are enriched on non-coding RNAs and thus drive the action of Sen1 to specific targets.3 Sen1 is a 5 to 3 helicase that can translocate on both single-stranded DNA and RNA.23,24 However, transcription termination involves exclusively the activity of Sen1 on the RNA component of the EC.24 The current model posits that, similar to Rho, Sen1 translocates along the nascent RNA to catch up with RNAPII and induce EC dissociation. X-ray crystal structures of the Escherichia coli RNA polymerase in complex with benzoxazinorifamycins. 6a, Supplementary Movie4). Accordingly, there was relatively little change in the torso (1A) domain and ATP-binding site, but the head (2A) domain moved away from the downstream side of RNAP (curved and straight orange arrows Fig. Crystal structure of a transcribing RNA Polymerase II complex reveals a complete transcription bubble, Mechanisms of bacterial transcription termination: all good things must end. T.N., M.M., and C.J.D. 2AFig. The plate was read using a Pherastar FS (BMG Labtech) using a 610nm excitation, 675nm emission optical module. In line with the key roles of TFIIH in transcription and DNA repair, protein database searches reveal that genes encoding the 10 TFIIH subunits are found in most eukaryotic genomes and that their sequences are highly conserved from yeast to human. Learn the definition of DNA helicase, then explore how it gains access to DNA, its role, and its function in the. We would like to thank Profs Nick Dixon and Rick Lewis for helpful comments on the manuscript, and members of our respective laboratories and those of Dr Libor Krasny and Prof Markus Wahl for discussions. J. Biol. However, structural analyses of bacterial ECs crystallized in the so-called elemental paused state have revealed several important differences relative to previously characterized ECs that are potentially relevant for termination. Grimm, C., Bartuli, J., Boettcher, B. et al. Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. & Cramer, P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS. Chem. Source data are provided with this paper. The frozen cell pellet was resuspended in 100ml HisA buffer (20mM KH2PO4 pH7.8, 500mM NaCl, 20mM imidazole) supplemented with EDTA-free protease inhibitor cocktail (1concentration S8830, Sigma-Aldrich) and 100l of 4mg/ml DNaseI (DN25, Sigma-Aldrich) at 4C. 1, 638653 (1994). Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand. Residues 610774 form a continuous SF1 2A domain, which is usually split by a 2B insertion in SF1 helicases, that represents the head of HelD. Rabatinova, A. et al. Therefore, the structure presented represents an elongation complex (EC) with non-specified nucleic acid sequence (i.e., the reconstructed density shows well-defined ribose-phosphate groups with an average of random base sequences). Fairman-Williams ME, Guenther U-P, Jankowsky E. Structure and mechanism of helicases and nucleic acid translocases, Transcription termination and the control of the transcriptome: why, where and how to stop. Biol. Sung P, et al. Crit Rev Biochem Mol Biol. 8). Despite nuclease treatment of the cell lysate, upon 3D reconstruction of the core structure, nucleic acid was clearly visible indicating that throughout the purification process the core enzyme remained tightly bound to nucleic acids which protected them from nuclease treatment (Supplementary Table1). Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Lane, W. J. Wiedermannova, J. et al. Cell 59, 133138 (2015). 2). Biol. 361, 634643 (2006). Elife 6, e25478 (2017). Commun. Finally, insertion of the SCA into the secondary channel would block NTP entry into the active site. 12, 16191623 (2002). Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. Would you like email updates of new search results? Broyles, S. S., Yuen, L., Shuman, S. & Moss, B. Purification of a factor required for transcription of vaccinia virus early genes. USA 114, E3032E3040 (2017). Gkhan Tolun or Peter J. Lewis. DNA Helicase - an overview | ScienceDirect Topics Helicase-like transcription factor (HLTF) is a central mediator of the DNA damage response and maintains genome stability by regressing stalled replication forks. Gupta, M. K. et al. J. Biol. CRISPR-associated DinG protein (CasDinG) is essential to type IV-A CRISPR function. Complete pathway for protein disulfide bond formation encoded by poxviruses. 2022 Aug 12;50(14):8008-8022. doi: 10.1093/nar/gkac583. 8, 91 (2008). In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Plasmid template for the MGA transcription assay consists of a construct containing three strong consensus promoters, Thermus VV1-2/D252, pGP31 from Bacillus phage SPO1 and LacUV5 that directed transcription of an array containing 12 direct repeats of the MGA sequence (5-GGATCCCGACTGGCGAGAGCCAGGTAACGAATGGATCCTAAAAAC-3) followed by an E. coli tRNA-trp terminator. aInstitut Jacques Monod, Centre Nationale pour la Recherche Scientifique (CNRS), UMR 7592, Universit Paris Diderot, Sorbonne Paris Cit, Paris, France, bUniversit Paris-Saclay, Gif sur Yvette, France. b The same view of the active site region of the RNAP-HelD complex with the SCA of HelD shown in red with the acidic D56 and D57 residues shown as sticks. 2B). In all cases RNAP release strictly depends on transcriptional pausing. PubMed Central Helicase Structure. (e) Local resolution mapped to the consensus reconstruction density iso-surface (only a mild B-factor sharpening of -10 2 was applied). Struct. Sci. The enigmatic helicase DHX9 and its association with the hallmarks of Screen. PubMedGoogle Scholar. The enlarged box on the right corresponds to the boxed region shown for the whole complex on the left. ISSN 1545-9985 (online) & Darst, S. A. Molecular evolution of multisubunit RNA polymerases: structural analysis. The red conformation of HelD is matched to the pale green conformation of RNAP, and the purple HelD with the yellow RNAP. https://doi.org/10.1038/s41594-021-00655-w. Get the most important science stories of the day, free in your inbox. Multiplexed protein-DNA cross-linking: scrunching in transcription start site selection. DNA and RNA Basics: Replication, Transcription, and Translation J. Struct. (d) Angular orientation plots referring to the reconstructions in (b). J. Mol. Direct removal of RNA polymerase barriers to replication by accessory replicative helicases. Approximately 60% of the data for both the RNAP HelD complex and the EC were collected at 20 stage tilt to compensate for the effects of preferred particle orientation. Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. A. Natl Acad. 361, 644658 (2006). The bridge-helix and trigger-loop, are dynamic structures that play a key role in the transcription cycle44; the entry of the SCA into the secondary channel causes partial folding of the open trigger-loop conformation observed in the EC structure and a major distortion of the bridge-helix that would sterically clash with the template DNA in the active site (Fig. (f) A depiction of the promoter-bound yeast XPB homologue SSL2 from the yeast PIC bound to TFIIH and core mediator (Schilbach et al., 2017) (PDB:5oqm) analogous to a. CAS Following refinement of the per-particle motion and CTF parameters, post-processing of the final reconstruction yielded a resolution of 3.36 as determined by the Gold-standard Fourier-Shell Correlation (FSC=0.143) criterion in RELION61. Try refreshing the page, or contact customer support. 3). Detailed examination of the elongation complex also revealed important features associated with mechanistic aspects of RNA synthesis. Broyles, S. S. & Moss, B. Homology between RNA polymerases of poxviruses, prokaryotes and eukaryotes: nucleotide sequence and transcriptional analysis of vaccinia virus genes encoding 147-kDa and 22-kDa subunits. The translocation module is flanked by an RNAP-interaction domain (RID) at the N-terminal side and by an autoinhibitory (AI) domain at the C-terminus that interacts with the UvrA-interaction module and locks the protein in a closed and inactive conformation.17 Binding of Mfd RID to the 1 subunit of stalled RNAP, triggers a conformational change in Mfd that results in the displacement of the AI domain from the UvrA-interaction module and consequently the activation of the Mfd translocase activity.18 Mfd then binds to approximately 25bp of dsDNA upstream of the stalled RNAP and translocates toward the RNAP, ultimately inducing EC dissociation.12 After the RNA and RNAP have been released from the DNA, Mfd continues translocating in the same direction with RNAP still attached to it to render accessible the DNA lesion and only when UvrA-UvrB is recruited, Mfd and RNAP quit the template DNA.19,20, A hypertranslocation model has been proposed to explain the mechanisms of EC dissociation by Mfd (Fig. 1a). For example, Fanconi anaemia. Anyone you share the following link with will be able to read this content: Sorry, a shareable link is not currently available for this article. Cell 179, 15371550 (2019). O.P. Areas used for local classification is encircled in magenta. Given that the ATP binding site was not accessible in all of the structures that are forcing open the DNA binding clamp of RNAP, gate opening may be an event that occurs on conformational change of the CA during nucleic acid release and RNAP recycling. Orange arrows indicate the movement of the juxtaposed region of HelD, and cyan arrows regions of RNAP. (d) Orientation plot referring to the consensus reconstruction in (b). 193, 112 (2016). Google Scholar. J. Mol. The functions of the RecQ-like genes are unknown; however, a growing body of evidence points to a function in restarting DNA replication after the replication fork has become stalled. Broyles, S. S. & Moss, B. Sedimentation of an RNA polymerase complex from vaccinia virus that specifically initiates and terminates transcription. Structural basis of poxvirus transcription: vaccinia RNA polymerase complexes. The first peak corresponded to Holoenzyme (~650mM NaCl), and the second, minor peak, to the HelD complex (~950mM NaCl). Two different concentrations of purified RNAP were prepared at 23.3mg/ml and 8.2mg/ml for storage. 3D variability analysis was performed solving for 3 conformational modes, and the results visualised in ChimeraX64. Open Access Elife 8, e40981 (2019). and JavaScript. government site. Bric, A., Radebaugh, C. A. The overall appearance of the protein is that of a torso and head (domains 1A and 2A, respectively) flanked by a pair of muscular arms (SCA and CA), giving it a rather thuggish appearance (Fig. Rocha, E. P. C. The replication-related organization of bacterial genomes. designed transcription scaffolds and acquired cryo-EM data. Nucleic Acids Res 43, 28292840 (2015). Google Scholar. USA 78, 187191 (1981). Methods 55, 94106 (2011). The first thing you notice is the button. Siitonen HA, Kopra O, Kriinen H, Haravuori H, Winter RM, Smnen AM, Peltonen L, Kestil M. Hum Mol Genet. Vassylyev, D. G., Vassylyeva, M. N., Perederina, A., Tahirov, T. H. & Artsimovitch, I. CryoEM maps have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/) under accession codes EMD-21921 (RNAP-HelD) and EMD-21920 (RNAP elongation complex). Thank you for visiting nature.com. Chem. (e) Cartoon model of VETFs and downstream DNA with superposed ideal B DNA in transparent grey is shown. Nat. (h) Pol II core PIC model (PDB 5IY6) in cartoon representation and oriented by superposition of the Pol II core polymerase with the core vRNAP of the vaccinia PIC in (g). Black dots represent data obtained using wild-type HelD, magenta dots, data obtained using HelD K239A. Subunit colouring is the same as in Fig. Cell 110, 4354 (2002). Bethesda, MD 20894, Web Policies The authors declare that they have no conflicts of interest with the contents of this article, MeSH FEBS Lett. Sen1 translocation towards the paused RNAPII leads to invasion of the RNA exit channel by the prong and subsequent destabilizing RNAPII conformational changes. Transcription Factor IIH - an overview | ScienceDirect Topics CAS J Biochem. Allosteric modulation of the RNA polymerase catalytic reaction is an essential component of transcription control by rifamycins. The contacts the CA makes with the clamp, open the DNA-binding and RNA-exit channels to enable dissociation of nucleic acids from the EC, possibly assisted by interaction of the DNA with the positively-charged patch on the CA of HelD. In these proteins the ring shape is also used to physically separate two DNA strands during unwinding via a steric exclusion mechanism 18., 19.. C.G. Biol. Kang, J. Y. et al. 5c). Peters JM, Mooney RA, Kuan PF, Rowland JL, Keles S, Landick R. Rho directs widespread termination of intragenic and stable RNA transcription, Structure of the Rho transcription terminator: mechanism of mRNA recognition and helicase loading, Structural insights into RNA-dependent ring closure and ATPase activation by the Rho termination factor. Nature 551, 653657 (2017). 2023 Apr 7;6(1):379. doi: 10.1038/s42003-023-04751-z. Natl Acad. Structure and mechanism of helicases and nucleic acid translocases. A full comprehension of the mechanism of transcription termination by the three factors described here necessitates a detailed characterization of all the protein-protein interactions and EC structural transitions taking part in the process. Kouba, T. et al. The native RNAP-HelD preparation also contained the RNAP subunit19 and showed it bound on the downstream side of RNAP in a concave space between the two , , and subunits (Fig. For many of them, these protein rearrangements result in the translocation of the helicase along the bound nucleic acid to eventually unwind duplexes or remove proteins associated with the nucleic acids. Helicases were originally defined as enzymes that use the binding and/or hydrolysis of the ATP to promote separation of the two strands of a duplex nucleic acid molecule.1,2 A more relaxed definition, however, includes also other structurally-related proteins that hydrolyze ATP in a nucleic acid-dependent manner without necessarily sharing the ability to dissociate a duplex nucleic acid (e.g. J. Biol. Biochem 76, 2350 (2007). 1d). Structure of a Novel DNA-binding Domain of Helicase-like Transcription Following dialysis in QA buffer (20mM Tris-HCl pH 7.8, 150mM NaCl, 10mM MgCl2, 1mM DTT) the sample was loaded onto a 1ml MonoQ column pre-equilibrated in TrisA. This website helped me pass! SARS-CoV-2 nsp13 Restricts Episomal DNA Transcription without - PubMed The left arrow indicates the subsequent 165 clockwise rotation of the complex to give the view in b. Following clarification by centrifugation, the overnight dialysate was loaded onto a 1ml MonoQ column pre-equilibrated in QA buffer. NCI CPTC Antibody Characterization Program. Science 372, eaba8490 (2021). (c) FSC plots for consensus refinement and the separaty bodies of the multibody (MB) refinement. Stelter, M., Acajjaoui, S., McSweeney, S. & Timmins, J. 1 with the addition of green, and HelD red. J. Biol. 7, 12911308 (1993). Yuen, L., Davison, A. J. and G.T. cloned genes, produced proteins/complexes and performed experiments. FOIA Bioinformatics 23, 10731079 (2007). ADS . Through confocal microscopy, the plant nuclear DNA helicase (PDH65) was shown to be localized within the dense fibrillar component of pea nucleoli in the regions around the rDNA transcription sites (Fig. 6b). Disclaimer. Cryo-EM of the complete vRNAP was carried out in the cryo-EM facility of the University of Wrzburg (DFG INST 93/903-1 FUGG). 8600 Rockville Pike RNA polymerase is the main transcription enzyme. Cryo-EM maps have been deposited with the Electron Microscopy Data Bank (EMDB) under accession nos. Soc. Although the existent mechanistic studies tend to present mechanical and allosteric models as alternatives, the complex process of transcription termination might be better explained by an integrative model in which the mechanical action of the helicase on the EC would be necessary, albeit not sufficient, for subsequent RNAP destabilizing rearrangements. Natl Acad. Keller, A. et al. DNA damage response; DNA helicase; DNA repair; DNA replication; fork reversal; nucleic acid enzymology; replication restart; stalled replication fork. As for the RNAP-HelD complex, the final refined map was then subjected to density modification and automated model-based based sharpening in Phenix62,63 (see Supplementary Fig. Croll, T. I. ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. Disordered regions of the template and non-template strand are shown as dotted lines. This separation, along with widening of the primary channel, facilitates RNA exit from the complex. 2001 Jan;27(1):113-6. doi: 10.1038/83673. (b) Local resolution mapped to the reconstruction density isosurface. 6b). Defects in helicases functioning in one or more of these proces Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD, https://doi.org/10.1038/s41467-020-20157-5. The salt-bridge and H-bond contacts the CA makes with the clamp are listed in Supplementary Table4, but the bulk of interactions are made by hydrophobic residues with little sequence conservation between even closely-related genera (Supplementary Table5, Supplementary Fig. Superposition with the nucleic acids from the EC show that this positively-charged patch is in a position where it could interact with the downstream dsDNA (Supplementary Fig. Rho Factor: Transcription Termination in Four Steps Thus, upon dissociation of HelD, the core RNAP would be competent for re-use in transcription, as seen in the transcription recycling assays in Fig. The dotted circles show the location of the ln5 insertion, binding site (b) and E696-G705 insertion (c), respectively. My textbook (WJEC Biology for AS level) says it is DNA helicase that breaks the hydrogen bonds while RNA polymerase catalyses the formation of bonds between RNA nucleotides. Natl Acad. The .gov means its official. Unauthorized use of these marks is strictly prohibited. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high . DNA Helicase: Definition, Role & Function - Study.com 2 DNA contacts in the PIC. Crystallogr 60, 21262132 (2004). Nature 505, 372377 (2014). 11, 1477 (2020). Nat Genet. All authors contributed to the analysis of the data and the interpretation of the results. The other arm pushes open the primary DNA-binding channel of RNAP, causing a conformational change that releases bound DNA. Role of Cockayne Syndrome Group B Protein in Replication Stress: Implications for Cancer Therapy. Sci. 6a; Supplementary Movie4). Genome Res. ), and NHMRC (GNT1184012 to G.T.). The conserved active site Asp residues that chelate the catalytic Mg2+ (green sphere, grey dashed lines) are also shown to illustrate how the tip of the SCA cages but does not interact directly with residues involved in RNAP catalysis. Senataxin (SETX) is a DNA-RNA helicase whose C-terminal region shows homology to the helicase domain of the yeast protein Sen1p. Genes Dev. Annu. It helped me pass my exam and the test questions are very similar to the practice quizzes on Study.com. Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. Commun. The elemental mechanism of transcriptional pausing. Keller, A. N. et al. At the same time, the 2 lobe moves up (straight cyan arrow, Fig. Newing, T.P., Oakley, A.J., Miller, M. et al. DNA Ligase Function & Role | Ligase in DNA Replication, DNA Polymerase | Definition, Structure & Function, DNA Polymerase & RNA Primase | Functions & End Products, Complementation Tests | Alleles, Loci & Crosses, DNA Replication Fork | Overview & Functions, DNA Replication Models | Semiconservative, Conservative & Dispersive. Crystal structure of a yeast TBP/TATA-box complex. DNase footprinting revealed that HLTF binds to the parental duplex ahead of the fork and at the leading edge behind the fork. Similarly, Rad26 (eukaryotic) and RapA (prokaryotic), Swi2/Snf2 family helicases also bind on the upstream side of RNAP and reverse-translocate stalled complexes during their reactivation15,16. 44, W344W350 (2016). RNA polymerase-induced remodelling of NusA produces a pause enhancement complex. Rho is a homo-hexameric SF5 RNA helicase that is responsible for roughly 20% of transcription termination events in E. coli.7 The first step of Rho-dependent termination is the recruitment of the helicase to the so-called rut (for Rho-utilization) sites in the nascent RNA.