scholarly journals Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription

2021 ◽  
Author(s):  
Benoit Arragain ◽  
Quentin Durieux Trouilleton ◽  
Florence Baudin ◽  
Stephen Cusack ◽  
Guy Schoehn ◽  
...  
Keyword(s):  
Active Site ◽  
Transcription Initiation ◽  
Conformational Dynamics ◽  
Replication Initiation ◽  
Genome Replication ◽  
La Crosse Virus ◽  
Future Design ◽  
Active Site Cavity ◽  
Life Threatening

Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize by cryo-electron microscopy its conformational dynamics, unveiling the precise molecular mechanics underlying its essential activities. Replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that firstly prompt primer cleavage and secondly promote primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The detailed uncovered mechanics will be crucial for future design of antivirals counteracting bunyaviral life-threatening pathogens.

scholarly journals Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

2021 ◽  
Author(s):  
Tomas Kouba ◽  
Dominik Vogel ◽  
Sigurdur R. Thorkelsson ◽  
Emmanuelle R. J. Quemin ◽  
Harry M. Williams ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Rna Synthesis ◽  
Antiviral Drug ◽  
Structural Data ◽  
Lassa Virus ◽  
Genome Replication ◽  
L Protein ◽  
Active Site Cavity

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

scholarly journals TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

2017 ◽  
Vol 114 (28) ◽  
pp. E5539-E5548 ◽  
Author(s):  
Saumya Gopalkrishnan ◽  
Wilma Ross ◽  
Albert Y. Chen ◽  
Richard L. Gourse
Keyword(s):  
Active Site ◽  
Transcription Initiation ◽  
Coiled Coil ◽  
Global Regulator ◽  
Secondary Channel ◽  
Global Regulators ◽  
Extrachromosomal Elements ◽  
Distant Homolog ◽  
Active Site Region

TheEscherichia coliF element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β′ rim helices that contribute to the ppGpp binding site in the DksA–ppGpp–RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements.

scholarly journals Crystal structures of hFPPS in complex with novel anticancer drug leads

2014 ◽  
Vol 70 (a1) ◽  
pp. C712-C712
Author(s):  
Jaeok Park ◽  
Chun Leung ◽  
Yih-Shyan Lin ◽  
Joris De Schutter ◽  
Youla Tsantrizos ◽  
...  
Keyword(s):  
Physicochemical Properties ◽  
Crystal Structures ◽  
Anticancer Agents ◽  
Active Site ◽  
Farnesyl Pyrophosphate ◽  
Allosteric Site ◽  
Cellular Processes ◽  
Inhibitory Site ◽  
Active Site Cavity

Human farnesyl pyrophosphate synthase (hFPPS) produces farnesyl pyrophosphate, an isoprenoid required for a variety of essential cellular processes. Inhibition of hFPPS has been well established as the mechanism of action of the nitrogen-containing bisphosphonate (N-BP) drugs, currently best known for their anti-bone resorptive effects. Recent investigations indicate that hFPPS inhibition also produces potent anticancer effects both in vitro and vivo: N-BPs inhibit proliferation, motility, and viability of tumor cells, and act in synergy with other anticancer agents [1,2]. However, the physicochemical properties of the current N-BP drugs seriously compromise their full anticancer potential in non-skeletal tissues. They show poor membrane permeability and extreme affinity to bone, due mainly to their highly charged bisphosphonate moiety, which mimics the pyrophosphate of the substrates of hFPPS. Both the substrates and N-BPs bind to hFPPS via Mg ion-mediated interactions between their pyrophosphate/bisphosphonate moiety and two aspartate-rich surfaces of the enzyme's active site cavity. Recently, we took a structure-guided approach to develop bisphosphonates with higher lipophilicity for enhanced uptake into non-skeletal tissues. Surprisingly, some of the new compounds were found to bind to hFPPS even in the absence of Mg ions. Crystal structures of hFPPS in complex with a representative compound revealed that this bisphosphonate binds to the enzyme's active site in the presence of Mg ions, but also to a nearby allosteric inhibitory site in their absence. Furthermore, removal of a phosphonate group from the bisphosphonate moiety of this compound resulted in an inhibitor that binds exclusively to the allosteric site. Based on the crystal structures with these lead compounds, we generated of a novel class of non-bisphosphonate, allosteric inhibitors of hFPPS with superior physicochemical properties than those of the current N-BP drugs for broader tissue distribution.

scholarly journals Set2-Dependent K36 Methylation Is Regulated by Novel Intratail Interactions within H3

2009 ◽  
Vol 29 (24) ◽  
pp. 6413-6426 ◽  
Author(s):  
James N. Psathas ◽  
Suting Zheng ◽  
Song Tan ◽  
Joseph C. Reese
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Transcription Initiation ◽  
Gene Activation ◽  
N Terminus

ABSTRACT Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

scholarly journals Characterization of the 5′ Ends for Polyadenylated RNAs Synthesized during the Replication of Hepatitis Delta Virus

1999 ◽  
Vol 73 (8) ◽  
pp. 6533-6539 ◽  
Author(s):  
Severin Gudima ◽  
Kate Dingle ◽  
Ting-Ting Wu ◽  
Gloria Moraleda ◽  
John Taylor
Keyword(s):  
Transcription Initiation ◽  
Hepatitis Delta Virus ◽  
Circular Rna ◽  
Genome Replication ◽  
Genomic Rna ◽  
Experimental Conditions ◽  
Hepatitis Delta ◽  
Polyadenylated Rnas ◽  
Delta Virus

ABSTRACT The genome of hepatitis delta virus (HDV) is a 1,679-nucleotide (nt) single-stranded circular RNA that is predicted to fold into an unbranched rodlike structure. During replication, two complementary RNAs are also detected: an exact complement, referred to as the antigenome, and an 800-nt polyadenylated RNA that could act as the mRNA for the delta antigen. We used a 5′ rapid amplification of cDNA ends procedure, followed by cloning and sequencing, to determine the 5′ ends of the polyadenylated RNAs produced during HDV genome replication following initiation under different experimental conditions. The analyzed RNAs were from the liver of an infected woodchuck and from a liver cell line at 6 days after transfection with either an HDV cDNA or ribonucleoprotein (RNP) complexes assembled in vitro with HDV genomic RNA and purified recombinant small delta protein. In all three situations the 5′ ends mapped specifically to nt 1630. In relationship to what is called the top end of the unbranched rodlike structure predicted for the genomic RNA template, this site is located 10 nt from the top, and in the middle of a 3-nt external bulge. Following transfection with RNP, such specific 5′ ends could be detected as early as 24 h. We next constructed a series of mutants of this predicted bulge region and of an adjacent 6-bp stem and the top 5-nt loop. Some of these mutations decreased the ability of the genome to undergo antigenomic RNA synthesis and accumulation and/or altered the location of the detected 5′ ends. The observed end located at nt 1630, and most of the novel 5′ ends, were consistent with transcription initiation events that preferentially used a purine. The present studies do not prove that the detected 5′ ends correspond to initiation sites and do not establish the hypothesis that there is a promoter element in the vicinity, but they do show that the location of the observed 5′ ends could be controlled by nucleotide sequences at and around nt 1630.

scholarly journals Structural basis of the complementary activity of two ketosynthases in aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Jiwon Yeon ◽  
Eunha Hwang ◽  
...  
Keyword(s):  
Active Site ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Structural Basis ◽  
Gram Negative Bacteria ◽  
Aryl Group ◽  
Vitro Assay ◽  
Active Site Cavity

AbstractAryl polyenes (APE) are one of the most widespread secondary metabolites among gram-negative bacteria. In Acinetobacter baumannii, strains belonging to the virulent global clone 2 (GC2) mostly contain APE biosynthesis genes; its relevance in elevated pathogenicity is of great interest. APE biosynthesis gene clusters harbor two ketosynthases (KSs): the heterodimeric KS-chain length factor complex, ApeO-ApeC, and the homodimeric ketoacyl-acyl carrier protein synthase I (FabB)-like KS, ApeR. The role of the two KSs in APE biosynthesis is unclear. We determined the crystal structures of the two KSs from a pathogenic A. baumannii strain. ApeO-ApeC and ApeR have similar cavity volumes; however, ApeR has a narrow cavity near the entrance. In vitro assay based on the absorption characteristics of polyene species indicated the generation of fully elongated polyene with only ApeO-ApeC, probably because of the funnel shaped active site cavity. However, adding ApeR to the reaction increases the throughput of APE biosynthesis. Mutagenesis at Tyr135 in the active site cavity of ApeR reduces the activity significantly, which suggests that the stacking of the aryl group between Tyr135 and Phe202 is important for substrate recognition. Therefore, the two KSs function complementarily in the generation of APE to enhance its production.

scholarly journals Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer

2005 ◽  
Vol 79 (1) ◽  
pp. 277-288 ◽  
Author(s):  
Todd C. Appleby ◽  
Hartmut Luecke ◽  
Jae Hoon Shim ◽  
Jim Z. Wu ◽  
I. Wayne Cheney ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Structural Information ◽  
Structural Features ◽  
Replication Initiation ◽  
Front Face ◽  
Large Cleft ◽  
Active Site Cavity

ABSTRACT Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

CONFORMATIONAL DYNAMICS OF HIV-1 PROTEASE: A COMPARATIVE MOLECULAR DYNAMICS SIMULATION STUDY WITH MULTIPLE AMBER FORCE FIELDS

2012 ◽  
Vol 10 (06) ◽  
pp. 1250018 ◽  
Author(s):  
BISWA RANJAN MEHER ◽  
MATTAPARTHI VENKATA SATISH KUMAR ◽  
SMRITI SHARMA ◽  
PRADIPTA BANDYOPADHYAY
Keyword(s):  
Force Field ◽  
Active Site ◽  
Force Fields ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Order Parameters ◽  
Dynamics Simulation ◽  
Active Site Cavity ◽  
Amber Force Fields ◽  
Hiv 1

Flap dynamics of HIV-1 protease (HIV-pr) controls the entry of inhibitors and substrates to the active site. Dynamical models from previous simulations are not all consistent with each other and not all are supported by the NMR results. In the present work, the effect of force field on the dynamics of HIV-pr is investigated by MD simulations using three AMBER force fields ff99, ff99SB, and ff03. The generalized order parameters for amide backbone are calculated from the three force fields and compared with the NMR S2 values. We found that the ff99SB and ff03 force field calculated order parameters agree reasonably well with the NMR S2 values, whereas ff99 calculated values deviate most from the NMR order parameters. Stereochemical geometry of protein models from each force field also agrees well with the remarks from NMR S2 values. However, between ff99SB and ff03, there are several differences, most notably in the loop regions. It is found that these loops are, in general, more flexible in the ff03 force field. This results in a larger active site cavity in the simulation with the ff03 force field. The effect of this difference in computer-aided drug design against flexible receptors is discussed.

scholarly journals Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome

2018 ◽  
Author(s):  
Emma Briggs ◽  
Graham Hamilton ◽  
Kathryn Crouch ◽  
Craig Lapsley ◽  
Richard McCulloch
Keyword(s):  
Transcription Initiation ◽  
Protozoan Parasite ◽  
Wide Distribution ◽  
Replication Initiation ◽  
Rrna Genes ◽  
Genome Replication ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Genome Wide ◽  
Intergenic Regions

AbstractR-loops are stable RNA-DNA hybrids that have been implicated in transcription initiation and termination, as well as in telomere homeostasis, chromatin formation, and genome replication and instability. RNA Polymerase (Pol) II transcription in the protozoan parasite Trypanosoma brucei is highly unusual: virtually all genes are co-transcribed from multigene transcription units, with mRNAs generated by linked trans-splicing and polyadenylation, and transcription initiation sites display no conserved promoter motifs. Here, we describe the genome-wide distribution of R-loops in wild type mammal-infective T. brucei and in mutants lacking RNase H1, revealing both conserved and diverged functions. Conserved localisation was found at centromeres, rRNA genes and retrotransposon-associated genes. RNA Pol II transcription initiation sites also displayed R-loops, suggesting a broadly conserved role despite the lack of promoter conservation or transcription initiation regulation. However, the most abundant sites of R-loop enrichment were within the intergenic regions of the multigene transcription units, where the hybrids coincide with sites of polyadenylation and nucleosome-depletion. Thus, instead of functioning in transcription termination, most T. brucei R-loops act in a novel role, promoting RNA Pol II movement or mRNA processing. Finally, we show there is little evidence for correlation between R-loop localisation and mapped sites of DNA replication initiation.

scholarly journals The enzymatic activity of the nsp14 exoribonuclease is critical for replication of Middle East respiratory syndrome-coronavirus

2020 ◽  
Author(s):  
Natacha S. Ogando ◽  
Jessika C. Zevenhoven-Dobbe ◽  
Clara C. Posthuma ◽  
Eric J. Snijder
Keyword(s):  
Middle East ◽  
Active Site ◽  
Rna Synthesis ◽  
Reverse Genetics ◽  
Antiviral Drug ◽  
Middle East Respiratory Syndrome ◽  
Genome Replication ◽  
Additional Function ◽  
Knockout Mutants

AbstractCoronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains an N-terminal 3’-to-5’ exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase) domain. While the latter presumably operates during viral mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. This is in agreement with observations previously made for alpha- and gammacoronaviruses. Only a single MERS-CoV ExoN active site mutant could be recovered, likely because the introduced D191E substitution is highly conservative in nature. For 11 other MERS-CoV ExoN active site mutants, not a trace of RNA synthesis could be detected, unless – in some cases – reversion had first occurred. Subsequently, we expressed and purified recombinant MERS-CoV nsp14 and established in vitro assays for both its ExoN and N7-MTase activities. All ExoN knockout mutations that were lethal when tested via reverse genetics were found to severely decrease ExoN activity, while not affecting N7-MTase activity. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV.ImportanceThe bifunctional nsp14 subunit of the coronavirus replicase contains 3’-to-5’ exoribonuclease (ExoN) and N7-methyltransferase (N7-MTase) domains. For the betacoronaviruses MHV and SARS-CoV, the ExoN domain was reported to promote the fidelity of genome replication, presumably by mediating some form of proofreading. For these viruses, ExoN knockout mutants are alive while displaying an increased mutation frequency. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication. Both enzymatic activities of (recombinant) MERS-CoV nsp14 were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

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