positive stranded viral RNA replication / host cell nucleolus / stringent response / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity ...positive stranded viral RNA replication / host cell nucleolus / stringent response / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / picornain 3C / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / T=pseudo3 icosahedral viral capsid / cytosolic ribosome assembly / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / host cell cytoplasmic vesicle membrane / maintenance of translational fidelity / response to radiation / ribosomal large subunit assembly / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / protein complex oligomerization / ribosome binding / monoatomic ion channel activity / ribosomal small subunit biogenesis / symbiont-mediated suppression of host gene expression / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / tRNA binding / cytoplasmic translation / RNA helicase activity / rRNA binding / negative regulation of translation / ribosome / RNA helicase / structural constituent of ribosome / ribonucleoprotein complex / symbiont entry into host cell / induction by virus of host autophagy / translation / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / response to antibiotic / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / negative regulation of DNA-templated transcription / DNA-templated transcription / mRNA binding / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / DNA binding / RNA binding / zinc ion binding / ATP binding / membrane / metal ion binding / cytoplasm / cytosol 類似検索 - 分子機能
Leader peptide, picornavirus / Viral leader polypeptide zinc finger / Virion protein N terminal domain / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 ...Leader peptide, picornavirus / Viral leader polypeptide zinc finger / Virion protein N terminal domain / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L11, bacterial-type / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L16 signature 1. / : / Picornavirales 3C/3C-like protease domain / Ribosomal protein L10 / Picornavirales 3C/3C-like protease domain profile. / Ribosomal protein L10P / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L17 signature. / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11, RNA binding domain / Ribosomal protein L11/L12 / Ribosomal protein L36 signature. / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L32p, bacterial type / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Picornavirus/Calicivirus coat protein / Ribosomal protein L6, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S19, bacterial-type / Ribosomal protein L20 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein S13, bacterial-type / Ribosomal protein L27 signature. / Ribosomal protein S20 類似検索 - ドメイン・相同性
50S ribosomal protein L10 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS13 ...50S ribosomal protein L10 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS21 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS12 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Genome polyprotein / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25 / Large ribosomal subunit protein bL31 類似検索 - 構成要素
ジャーナル: Nat Commun / 年: 2021 タイトル: Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch. 著者: Chris H Hill / Lukas Pekarek / Sawsan Napthine / Anuja Kibe / Andrew E Firth / Stephen C Graham / Neva Caliskan / Ian Brierley / 要旨: Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we ...Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
全体 : Initiated 70S ribosome in complex with 2A protein from encephalom...
全体
名称: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV)
要素
複合体: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV)
タンパク質・ペプチド: 50S ribosomal protein L2
タンパク質・ペプチド: 50S ribosomal protein L3
タンパク質・ペプチド: 50S ribosomal protein L4
タンパク質・ペプチド: 50S ribosomal protein L5
タンパク質・ペプチド: 50S ribosomal protein L6
タンパク質・ペプチド: 50S ribosomal protein L9
タンパク質・ペプチド: 50S ribosomal protein L10
タンパク質・ペプチド: 50S ribosomal protein L11
タンパク質・ペプチド: 50S ribosomal protein L13
タンパク質・ペプチド: 50S ribosomal protein L14
タンパク質・ペプチド: 50S ribosomal protein L15
タンパク質・ペプチド: 50S ribosomal protein L16
タンパク質・ペプチド: 50S ribosomal protein L17
タンパク質・ペプチド: 50S ribosomal protein L18
タンパク質・ペプチド: 50S ribosomal protein L19
タンパク質・ペプチド: 50S ribosomal protein L20
タンパク質・ペプチド: 50S ribosomal protein L21
タンパク質・ペプチド: 50S ribosomal protein L22
タンパク質・ペプチド: 50S ribosomal protein L23
タンパク質・ペプチド: 50S ribosomal protein L24
タンパク質・ペプチド: 50S ribosomal protein L25
タンパク質・ペプチド: 50S ribosomal protein L27
タンパク質・ペプチド: 50S ribosomal protein L28
タンパク質・ペプチド: 50S ribosomal protein L29
タンパク質・ペプチド: 50S ribosomal protein L30
RNA: 23S ribosomal RNA
RNA: 16S ribosomal RNA
RNA: 5S ribosomal RNA
タンパク質・ペプチド: 50S ribosomal protein L31
タンパク質・ペプチド: 50S ribosomal protein L32
タンパク質・ペプチド: 50S ribosomal protein L33
タンパク質・ペプチド: 50S ribosomal protein L34
タンパク質・ペプチド: 50S ribosomal protein L35
タンパク質・ペプチド: 50S ribosomal protein L36
タンパク質・ペプチド: 30S ribosomal protein S2
タンパク質・ペプチド: 30S ribosomal protein S3
タンパク質・ペプチド: 30S ribosomal protein S4
タンパク質・ペプチド: 30S ribosomal protein S5
タンパク質・ペプチド: 30S ribosomal protein S6
タンパク質・ペプチド: 30S ribosomal protein S7
タンパク質・ペプチド: 30S ribosomal protein S8
タンパク質・ペプチド: 30S ribosomal protein S9
タンパク質・ペプチド: 30S ribosomal protein S10
タンパク質・ペプチド: 30S ribosomal protein S11
タンパク質・ペプチド: 30S ribosomal protein S12
タンパク質・ペプチド: 30S ribosomal protein S13
タンパク質・ペプチド: 30S ribosomal protein S14
タンパク質・ペプチド: 30S ribosomal protein S15
タンパク質・ペプチド: 30S ribosomal protein S16
タンパク質・ペプチド: 30S ribosomal protein S17
タンパク質・ペプチド: 30S ribosomal protein S18
タンパク質・ペプチド: 30S ribosomal protein S19
タンパク質・ペプチド: 30S ribosomal protein S20
タンパク質・ペプチド: 30S ribosomal protein S21
RNA: mRNA
タンパク質・ペプチド: Protein 2A
RNA: fMet-NH-tRNA(fMet)
リガンド: MAGNESIUM ION
リガンド: N-FORMYLMETHIONINE
リガンド: ZINC ION
リガンド: water
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超分子 #1: Initiated 70S ribosome in complex with 2A protein from encephalom...
超分子
名称: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV) タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1-#28, #30-#57 詳細: 70S ribosome subunits and initiator fMet-tRNA purified from E.coli. mRNA template was generated by in vitro transcription EMCV 2A protein was recombinantly expressed in E.coli
名称: mRNA / タイプ: rna / ID: 56 詳細: EMCV frameshift site flanked by the bacterial 5' UTR with Shine-Dalgarno sequence and 18 nt downstream region of the putative structure コピー数: 1
詳細: Initiated 70S ribosomes in 50 mM Tris-HCl pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2 were diluted tenfold into 20 mM HEPES pH 7.5, 100 mM potassium acetate, 1.5 mM MgCl2, 2.0 mM DTT. 2A ...詳細: Initiated 70S ribosomes in 50 mM Tris-HCl pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2 were diluted tenfold into 20 mM HEPES pH 7.5, 100 mM potassium acetate, 1.5 mM MgCl2, 2.0 mM DTT. 2A protein was dialysed (3K MWCO, 277K, 16 h) into the same buffer. Crosslinking reactions of 50 microliters comprising 75 nM ribosomes, 3.0 micromolar 2A and 2.0 mM bis(sulfosuccinimidyl)suberate (BS3) were performed on ice (30 min) immediately prior to grid preparation.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: Quantifoil R 2/2 400-mesh copper supports were coated with an additional ~ 60 angstrom layer of amorphous, evaporated carbon by flotation and thoroughly dried before use. Grids were made ...詳細: Quantifoil R 2/2 400-mesh copper supports were coated with an additional ~ 60 angstrom layer of amorphous, evaporated carbon by flotation and thoroughly dried before use. Grids were made hydrophilic by glow-discharge in air for 30 s. Three microliters of crosslinking reaction was applied to grids which were then blotted for 4.5 s and vitrified by plunging into liquid ethane using a Vitrobot MK IV (FEI) at 277K, 100% relative humidity..
詳細
75 nM ribosomes, 3.0 micromolar 2A crosslinked with 2.0 mM bis(sulfosuccinimidyl)suberate (BS3)
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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
撮影
フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 検出モード: INTEGRATING / 撮影したグリッド数: 1 / 実像数: 5730 / 平均露光時間: 0.59 sec. / 平均電子線量: 54.4 e/Å2 詳細: Images were collected as 23-frame movies. Autofocus every 10 micrometres, no drift measurement, 7 sec delay after stage shift and 3 sec delay after image shift
Movie frames were aligned and a dose-weighted average calculated with MotionCor2
粒子像選択
選択した数: 820475 詳細: Reference-free autopicking of particles was performed using the Laplacian-of-Gaussian function in Relion (200 - 250 angstrom diameter)
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1)
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 3.1)
最終 3次元分類
クラス数: 6 / 平均メンバー数/クラス: 20124 / ソフトウェア - 名称: RELION (ver. 3.1) 詳細: Focussed classification with signal subtraction and local angular searches was performed to separate particles based on 2A occupancy at the factor binding site