著者は、優れたテクニカル サポート メッテ Warrer をありがとうございます。この作業は、独立した研究のためデンマーク評議会によって支えられた |自然科学 [1323-00343B] と [DNRF120] デンマーク国立研究財団。

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charging+levels+of+four+tRNA+species+in+Escherichia+coli+Rel(+)+and+Rel(-)+strains+during+amino+acid+starvation:+a+simple+model+for+the+effect+of+ppGpp+on+translational+accuracy.">Charging levels of four tRNA species in Escherichia coli Rel(+) and Rel(-) strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy.</a> J Mol Biol. 307 (3), 785-798 (2001).</li><li>Kr&#252;ger, M. K., S&#248;rensen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoacylation+of+hypomodified+tRNAGlu+in+vivo.">Aminoacylation of hypomodified tRNAGlu in vivo.</a> J. Mol. Biol. 284 (3), 609-620 (1998).</li><li>Svenningsen, S. L., Kongstad, M., Stenum, T. S., Mu&#241;oz-G&#243;mez, A. J., S&#248;rensen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNA+is+highly+unstable+during+early+amino+acid+starvation+in+Escherichia+coli.">Transfer RNA is highly unstable during early amino acid starvation in Escherichia coli.</a> Nucleic Acids Res. 45 (2), 793-804 (2017).</li><li>Enriquez, J. A., Attardi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+aminoacylation-induced+conformational+changes+in+human+mitochondrial+tRNAs.">Evidence for aminoacylation-induced conformational changes in human mitochondrial tRNAs.</a> Proc Natl Acad Sci U S A. 93 (16), 8300-8305 (1996).</li><li>Parker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Errors+and+alternatives+in+reading+the+universal+genetic+code.">Errors and alternatives in reading the universal genetic code.</a> Microbiol Rev. 53 (3), 273-298 (1989).</li><li>Traxler, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global,+ppGpp-mediated+stringent+response+to+amino+acid+starvation+in+Escherichia+coli.">The global, ppGpp-mediated stringent response to amino acid starvation in Escherichia coli.</a> Mol Microbiol. 68 (5), 1128-1148 (2008).</li><li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+RNAs+Mediate+the+Rapid+Adaptation+of+Escherichia+coli+to+Oxidative+Stress.">Transfer RNAs Mediate the Rapid Adaptation of Escherichia coli to Oxidative Stress.</a> PLoS Genet. 11 (6), e1005302 (2015).</li><li>Kane, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+rare+codon+clusters+on+high-level+expression+of+heterologous+proteins+in+Escherichia+coli.">Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli.</a> Curr Opin Biotechnol. 6 (5), 494-500 (1995).</li><li>Baca, A. M., Hol, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+codon+bias:+a+method+for+high-level+overexpression+of+Plasmodium+and+other+AT-rich+parasite+genes+in+Escherichia+coli.">Overcoming codon bias: a method for high-level overexpression of Plasmodium and other AT-rich parasite genes in Escherichia coli.</a> Int J Parasitol. 30 (2), 113-118 (2000).</li><li>Li, S., Pelka, H., Schulman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anticodon+and+discriminator+base+are+important+for+aminoacylation+of+Escherichia+coli+tRNA+(Asn).">The anticodon and discriminator base are important for aminoacylation of Escherichia coli tRNA (Asn).</a> J Biol Chem. 268 (24), 18335-18339 (1993).</li><li>Rizzino, A. A., Freundlich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+in+vivo+aminoacylation+by+periodate+oxidation:+tRNA+alterations+and+iodate+inhibition.">Estimation of in vivo aminoacylation by periodate oxidation: tRNA alterations and iodate inhibition.</a> Anal Biochem. 66 (2), 446-449 (1975).</li><li>Dittmar, K. A., Sorensen, M. A., Elf, J., Ehrenberg, M., Pan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+charging+of+tRNA+isoacceptors+induced+by+amino-acid+starvation.">Selective charging of tRNA isoacceptors induced by amino-acid starvation.</a> EMBO Rep. 6 (2), 151-157 (2005).</li><li>Zhou, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+reference+genes+for+quantifying+transcriptional+responses+of+Escherichia+coli+to+protein+overexpression+by+quantitative+PCR.">Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR.</a> BMC Mol Biol. 12 (1), 18 (2011).</li><li>Jacobson, A., Gillespie, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+events+occurring+during+recovery+from+prolonged+glucose+starvation+in+Escherichia+coli.">Metabolic events occurring during recovery from prolonged glucose starvation in Escherichia coli.</a> J Bacteriol. 95 (3), 1030-1039 (1968).</li><li>McCarthy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+magnesium+starvation+on+the+ribosome+content+of+Escherichia+coli.">The effects of magnesium starvation on the ribosome content of Escherichia coli.</a> Biochim Biophys Acta (BBA)-Specialized Section on Nucleic Acids and Related Subjects. 55 (6), 880-889 (1962).</li><li>Zundel, M. A., Basturea, G. N., Deutscher, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+ribosome+degradation+during+starvation+in+Escherichia+coli.">Initiation of ribosome degradation during starvation in Escherichia coli.</a> Rna. 15 (5), 977-983 (2009).</li><li>Piir, K., Paier, A., Liiv, A., Tenson, T., Maivali, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+degradation+in+growing+bacteria.">Ribosome degradation in growing bacteria.</a> EMBO Rep. 12 (5), 458-462 (2011).</li><li>Schaechter, M., Maaloe, O., Kjeldgaard, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependency+on+medium+and+temperature+of+cell+size+and+chemical+composition+during+balanced+grown+of+Salmonella+typhimurium.">Dependency on medium and temperature of cell size and chemical composition during balanced grown of Salmonella typhimurium.</a> J Gen Microbiol. 19 (3), 592-606 (1958).</li><li>Neidhardt, F. C., Bloch, P. L., Smith, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+medium+for+enterobacteria.">Culture medium for enterobacteria.</a> J Bacteriol. 119 (3), 736-747 (1974).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (1989).</li><li>Sorensen, M. A., Jensen, K. F., Pedersen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concentrations+of+ppGpp+decrease+the+RNA+chain+growth+rate.+Implications+for+protein+synthesis+and+translational+fidelity+during+amino+acid+starvation+in+Escherichia+coli.">High concentrations of ppGpp decrease the RNA chain growth rate. Implications for protein synthesis and translational fidelity during amino acid starvation in Escherichia coli.</a> J Mol Biol. 236 (2), 441-454 (1994).</li><li>Tian, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Curtailing+of+the+Stringent+Response+by+Toxin-Antitoxin+Module-Encoded+mRNases.">Rapid Curtailing of the Stringent Response by Toxin-Antitoxin Module-Encoded mRNases.</a> J Bacteriol. 198 (14), 1918-1926 (2016).</li></ol>

著者が明らかに何もありません。

このプロトコルは、特定の大腸菌の充電レベルを同時に測定する方法をについて説明します Trna と異なったサンプルの Trna の相対的なレベルを比較します。1) 携帯電話の tRNA 充電レベルが保持されます、tRNA は、異なったサンプルの相対的な tRNA のレベルを確実に比較できる、そのような方法で数量を正規化するには 2) と 3) sp を確実にこのような方法でサンプルを処理するプロトコル...

以下では、特定の Trna の定量化手法を提案し、北にしみが付くことによってレベルに充電を測定します。最初 Varshneyらが開発した技術に基づく1。 トリクロロ酢酸 (TCA) に細胞を収穫し、RNA 精製中の 0 の ° C でサンプルを維持する、tRNA とアミノ酸のエステル結合が保存された2,3,4。Aminoacylated Trna で区別できる nonacylated 対応ゲル電気泳動とブロッティング、ため、aminoacylated の減少移動する共有結合アミノ酸5によるゲルの tRNA。さらに、使用頻度の低い tRNAselC 4を過剰発現細胞のスパイクの添加 tRNA の量の正規化のためのプロトコルを提案する.
細菌の細胞の翻訳機械の絶対に不可欠な部分の最も豊富な分子である Trna。Trna はアミノ酸をバインドし、翻訳のリボゾームにそれらを転送します。Trna のアミノアシル化 (充電) するためにアミノ酸の結合は、アミノアシル tRNA シンテターゼによって促進されます。別の充電された Trna の相対的な豊かさはタンパク質合成の忠実性を確保するため重要な同系の過少請求指定されたメッセンジャー RNA (mRNA) コドンの tRNA 同系近く tRNA がされる可能性が高くなりますので誤ってそのアミノ酸をリボソーム6成長ポリペプチド鎖に配信します。充電された tRNA の重要性は、tRNA; の充電レベルの深刻な低下にエシェリヒア属大腸菌細胞の広範な応答に反映されます。厳格な応答です。厳格な応答時に Trna、リボソーム Rna とほとんど Mrna の合成はアミノ酸の生合成やストレスの生存に関連付けられている特定の Mrna の転写を支持して低下され、細胞の成長率を劇的に下げた7.さらに、われわれや他の最近の作品は、大腸菌が tRNA レベルの調整可能性があることを示唆している制限する翻訳4,8ストレスへの応答で、tRNA の大半を積極的に劣化することを示しています。このようなストレスに対処するために重要です。したがって、tRNA 微量含有量と充電レベルの信頼性の高い計測は完全に細菌のストレス反応を理解するための重要なツールになります。
エシェリヒア属大腸菌組換え蛋白質を表現する使用され、種のコドン使用の違い、最適な表現はしばしば直面する問題9。これは組換え mRNA10を変換に必要な追加の Trna の式によって回避できます。TRNA のような株のレベルを充電の測定ことができるトラブルシューティングの努力を導くし、蛋白質の表現を最適化するため。
このメソッドは、また"mischarging"の検出を可能にします。非同種のアミノ酸を tRNA 分子 aminoacylated。種類のアミノ酸によってアミノアシル化は、ポリアクリルアミドゲル1,11をわずかに異なる速度で移行する tRNA を引き起こす可能性があります。いくつかのケースでは、そうでなければ同じ Trna3異なる修飾パターンを区別するためにメソッドを使用できます。
別は、tRNA の充電レベルの調査は酸化を過ヨウ素酸の生化学的なプロシージャを設立しました。メソッドは、tRNA から保護 aminoacylated に酸化が過ヨウ素酸中性 tRNA ではない観察に依存します。後、酸化処理収穫された RNA の充電レベルの見積もりに使用、tRNA の充電過ヨウ素酸します。しかし、このようにいくつかの不正確さ12を提供する治療によって影響を受けるいくつかの Trna の充電を示されています。メソッドは、浄化された RNA から直接充電、化学か酵素の反応からどんな先入観を排除対策を紹介しました。このメソッドの制限の 1 つは時間がかかり、やや多く Trna のデータを収集するは面倒だが、同じ北のしみの除去および複数の Trna の reprobed できるように、時だけ 1 つの tRNA 種が検出されました。
互いにサンプルの正規化の信頼性の高い方法は、異なるサンプルの間で分子の相対的なレベルを比較することの研究で不可欠です。ここまれな tRNAselCを過剰発現大腸菌細胞の小さい因数のスパイクでとしての RNA の浄化の前にすべての実験サンプルに追加されます RNA の正規化の手順を紹介します。このメソッドは、Trna を調べるときだけでなく、RNA の任意の種の相対的な定量化のためのすべてのサンプルで同じ細胞濃度で提示実験のセットアップがこのような内因性 RNA がないする信頼できる便利です。たとえば、「ハウスキーピング」RNA のようなリボソーム RNA レベルは別の RNA 間の相対的な量を比較する内因性参照サンプル13、によく使用されます。しかし、これは少し使用の参照 RNA の細胞濃度のサンプル間の相違ストレス応答15,16,17中または中にサンプリングが発生した場合、リボソーム RNA のためのケースをすることができます。固定相17へ参入。スパイクで細胞の RNA の浄化の前に実験サンプルへの添加は、正規化サンプリング設定の独立の堅実で正確な方法を提供します。標準化の別の方法は、RNA 精製後 1 つまたは複数のスパイクで Rna の実験サンプルに追加です。ただし、このメソッドは、サンプルの RNA の回復の違いについては考慮しません。
エシェリヒア属大腸菌細胞を用いた大腸菌の実験でスパイクの細胞 (図 1を参照) として参照 RNA が欠点があるその過剰さらに外因性大腸菌の少量の合計する RNA、サンプル。我々 はスパイクでのみセル (参照図 2,の車線「selC」というラベルの付いたしみ北) 実験サンプルと並行を含むサンプルを分析することによりこのほかの修正します。提示プロトコルエシェリヒア属大腸菌K-12 の開発がほとんどの細菌種に適用する可能性があります。

<table><tbody><tr><td>Urea</td><td>Merck</td><td>57-13-6</td><td>Purity &gt;99%</td></tr><tr><td>Polyacrylamide</td><td>Serva</td><td>79-06-7</td><td></td></tr><tr><td>Bis (N,N&#039;-Methylene-bis-acrylamide)</td><td>BIO-RAD</td><td>161-0201</td><td></td></tr><tr><td>Trichloroacetic acid (TCA)</td><td>Sigma</td><td>76-03-9</td><td></td></tr><tr><td>Phenol</td><td>Merck</td><td>108-95-2</td><td></td></tr><tr><td>IPTG</td><td></td><td></td><td></td></tr><tr><td>Glucose</td><td></td><td></td><td></td></tr><tr><td>Sodium Acetate</td><td></td><td></td><td></td></tr><tr><td>EDTA</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td></td><td></td><td></td></tr><tr><td>Tris</td><td></td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td></td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td></td><td></td><td></td></tr><tr><td>NaH2PO4</td><td></td><td></td><td></td></tr><tr><td>Sodium Citrate</td><td></td><td></td><td></td></tr><tr><td>SDS</td><td></td><td></td><td></td></tr><tr><td>Herring Sperm DNA</td><td>Sigma</td><td>100403-24-5</td><td></td></tr><tr><td>BSA</td><td></td><td></td><td></td></tr><tr><td>Polivinylpyrrolidone</td><td></td><td></td><td></td></tr><tr><td>Ficoll</td><td></td><td></td><td></td></tr><tr><td>P32 &amp;gamma;ATP</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>DNA oligos (probes)</td><td>TAG Copenhagen</td><td></td><td></td></tr><tr><td>Hybond N&lt;sup&gt;+&lt;/sup&gt; membrane</td><td>GE Healthcare</td><td>RPN203B</td><td></td></tr><tr><td>Crosslinker</td><td></td><td></td><td></td></tr><tr><td>Electroblotter</td><td>BIO-RAD</td><td></td><td></td></tr><tr><td>Typhoon FLA 7000 Scanner</td><td>GE Healthcare</td><td>28955809</td><td></td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td></td></tr><tr><td>Hydridization oven</td><td></td><td></td><td></td></tr><tr><td>Geiger-M&amp;uuml;ller tube</td><td></td><td></td><td></td></tr><tr><td>Phosphor imager screen</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Hybridization tube</td><td></td><td></td><td></td></tr><tr><td>Culture Flasks</td><td></td><td></td><td></td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>20 ml centrifuge tubes</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ImageQuant</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

quantification of the abundance and charging levels of transfer rnas in escherichia coli

トランスファー RNA (tRNA) は、あらゆる有機体の並進機械の重要な部分です。Trna はバインドし、アミノ酸をリボソーム翻訳に転送します。別の Trna の相対的なレベルと aminoacylated の比を合計 tRNA、充電レベルとして知られている tRNA が精度と翻訳のスピードを決める重要な要素。したがって、豊かさと Trna の充電レベルは、たとえば種々 の応力条件下でのタンパク質合成の勉強を測定する重要な変数です。ここでは、tRNA を収穫し、相対的な豊かさと大腸菌における特定の tRNA 種の絶対的な充電レベルを直接測定する方法をについて説明します。TRNA は、tRNA とアミノ酸の間不安定な結合が維持されることそのような方法で収穫されます。RNA ゲル電気泳動および北しみが付くこと、充電、非荷電の Trna の分離の結果にさらされます。正規化のためのスパイクの細胞のための異なるサンプルで特定の Trna のレベルを比較できます。RNA 精製の前に、各サンプルにまれな tRNAselCの過剰大腸菌細胞の 5% を追加します。サンプルの興味の tRNA 種の量は、同じサンプルの tRNAselCの量に正規化されます。スパイクで細胞の RNA の浄化前の追加サンプルのセル換散の効率の違いも占める浄化されたスパイクで Rna のさらに利点があります。

豊かさと充電前に、とアミノ酸飢餓の中に 3 つの Trna のレベルがエシェリヒア属大腸菌K-12 で測定されたここで説明手順を使用しています。
アルギニン auxotroph ひずみ NF9154 (木ルー彼くっティ mtl supE44) 0.4% グリセロール、50 μ g/mL スレオニン、ロイシン、アルギニン、37 ° c で揺れ 5 μ G/ml ヒ?...

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Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

豊かさと充電転送エシェリヒア属大腸菌の Rna のレベルの定量化

スパイクの追加に基づく異なるサンプル間転移 RNA の相対的なレベルまたは他の短い RNA を比較する方法と同様、精製されたエシェリヒア属大腸菌RNA からレベルを充電転送の RNA を直接測定法を紹介します。参照の遺伝子を発現する細胞。

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

quantification-abundance-charging-levels-transfer-rnas-escherichia

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

9.3K ビュー.  University of Copenhagen. スパイクの追加に基づく異なるサンプル間転移 RNA の相対的なレベルまたは他の短い RNA を比較する方法と同様、精製されたエシェリヒア属大腸菌RNA からレベルを充電転送の RNA を直接測定法を紹介します。参照の遺伝子を発現する細胞。

ビデオ: Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 &#176;C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the N6-threonylcarbamoyladenosine TcdA Function

A protocol is presented for the production of tRNA(UUU) and the analysis of tRNA(UUU) in complex with the enzyme TcdA by agarose gel retardation assays.

タンパク質分析のためのタンパク質tRNAアガロースゲルリターデーションアッセイ<em&gt; N</em<sup&gt; 6</sup&gt; - スルホニルカルバモイルアデノシンTcdA機能

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of ...

生化学

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

リアルタイムPCRによる沿岸堆積物および定量16SのrRNA遺伝子と転写物からの同時DNA-RNA抽出

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

環境学

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

遺伝学

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

代謝ラベリングと Macroarrays を使用して転移 Rna のプロファイリング

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

細菌におけるタンパク質-RNA結合を定量するためのアッセイ

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

AQRNA-seqを用いたtRNA動態の解析

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

著者スポットライト:低分子RNAのマッピングとタンパク質翻訳メカニズムの解明におけるAQRNA-seqの役割

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

低分子RNAの定量のためのAQRNA-seq

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

細菌におけるRIBO-seq:NGSシーケンシングのためのサンプル収集とライブラリ調製プロトコル

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

マイクロアレイを用いたtRNAのアミノアシル化（充電）レベルのゲノムワイド解析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

豊かさと充電転送エシェリヒア属大腸菌の Rna のレベルの定量化

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タンパク質分析のためのタンパク質tRNAアガロースゲルリターデーションアッセイ

リアルタイムPCRによる沿岸堆積物および定量16SのrRNA遺伝子と転写物からの同時DNA-RNA抽出

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

代謝ラベリングと Macroarrays を使用して転移 Rna のプロファイリング

細菌におけるタンパク質-RNA結合を定量するためのアッセイ

低分子RNAの定量のためのAQRNA-seq

低分子RNAの定量のためのAQRNA-seq

低分子RNAの定量のためのAQRNA-seq

細菌におけるRIBO-seq:NGSシーケンシングのためのサンプル収集とライブラリ調製プロトコル

マイクロアレイを用いたtRNAのアミノアシル化（充電）レベルのゲノムワイド解析