Gli autori ringraziano Marit Warrer per l'eccellente assistenza tecnica. Questo lavoro è stato supportato dal Consiglio danese per la ricerca indipendente | Scienze naturali [1323-00343B] e la Fondazione di ricerca nazionale danese [DNRF120].

<ol>
	<li>Varshney, U., Lee, C. -. P., RajBhandary, U. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+aminoacylation+levels+of+tRNAs+in+vivo.+Application+to+studying+recognition+of+Escherichia+coli+initiator+tRNA+mutants+by+glutaminyl-tRNA+synthetase.">Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase.</a> J Biol Chem. 266 (36), 24712-24718 (1991).</li><li>Sorensen, 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=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>

Gli autori non hanno nulla a rivelare.

Questo protocollo descrive come misurare contemporaneamente il livello di carico delle specifiche Escherichia coli tRNAs e confrontare i livelli relativi dei tRNAs in diversi campioni. I punti critici del protocollo sono 1) per gestire i campioni in modo che i livelli di carica tRNA cellulare vengono mantenuti, 2) per normalizzare la quantità di tRNA in modo tale che i livelli relativi di tRNA in diversi campioni possono essere paragonati in modo affidabile e 3) per garantire la sp ecificity delle sonde selezio...

Di seguito, presentiamo un metodo per quantificare specifico tRNAs e misurare la loro carica livelli mediante Northern blotting. Il metodo si basa su una tecnica sviluppata da Varshney et al. 1. raccolta delle cellule in acido tricloroacetico (TCA) e mantenere i campioni a 0 ° C durante la purificazione di RNA, il legame estere tra il tRNA e l'aminoacido è conservata2,3,4. Aminoacylated tRNAs si distinguono dalle loro controparti nonacylated gel elettroforesi e macchiare nordico, dovuto ad una mobilità in diminuzione del aminoacylated tRNA nel gel, causato dalla covalentemente aminoacido5. Inoltre, vi presentiamo un protocollo per la normalizzazione della quantità di tRNA con aggiunta di spike-in cellule che overexpressing il raramente usato tRNAselC 4.
tRNAs sono alcune delle molecole più abbondanti nella cellula batterica e una parte vitale dell'apparato di traduzione. tRNAs legano gli amminoacidi e trasferirli i ribosomi traduzioni. L'associazione di aminoacidi per tRNAs (aminoacylation o ricarica) è facilitato dalla sintetasi del tRNA di aminoacyl. L'abbondanza relativa di diversi tRNAs carica è importante per assicurare la fedeltà della sintesi proteica, poiché sottorappresentanza delle cognate carica che tRNA per un codone determinato RNA messaggero (mRNA) aumenta la probabilità che un tRNA vicino cognate sarà erroneamente consegnare suo aminoacido a catena del polipeptide crescente sul ribosoma6. L'importanza della carica tRNA si riflette nella vasta risposta della cella e. coli a un drastico calo dei livelli di carica di un tRNA; la risposta rigorosa. Durante la risposta rigorosa la sintesi di tRNA, RNA ribosomiale (rRNA) e maggior parte dei mRNAs è abbassata a favore di trascrizione di mRNA specifici connessi con la sopravvivenza dell'amminoacido biosintesi e stress, e il tasso di crescita delle cellule è abbassato drasticamente7 . Inoltre, il recente lavoro da noi e gli altri ha mostrato che e. coli degrada attivamente la maggior parte dei suoi tRNA in risposta alle sollecitazioni che limitano traduzione4,8, suggerendo che la regolazione dei livelli di tRNA può essere importante per far fronte a tali sollecitazioni. Così, misurazioni affidabili delle abbondanze di tRNA e livelli di ricarica sarà uno strumento importante per comprendere pienamente le risposte batteriche di sforzo.
E. coli è comunemente usato per esprimere la proteina ricombinante e a causa delle differenze nell'uso di codone tra specie, espressione non ottimale è un problema spesso affrontato9. Questo può essere aggirato da espressione di tRNAs aggiuntive necessarie per tradurre il mRNA ricombinante10. Misurazioni di tRNA livelli in tali ceppi di ricarica potrebbero guidare gli sforzi sulla risoluzione dei problemi e ottimizzare l'espressione della proteina.
Questo metodo consente anche la rilevazione di "orifizio"; una molecola di tRNA aminoacylated con un aminoacido non-affine. Aminoacylation di aminoacidi diversi può causare un tRNA per la migrazione con velocità leggermente diverse attraverso i gel di poliacrilammide1,11. In alcuni casi, il metodo utilizzabile anche per distinguere diversi modifica modelli altrimenti identici tRNAs3.
Un altro istituita procedura biochimica per l'indagine di tRNA livelli di carica è periodato ossidazione. Il metodo si basa sull'osservazione che periodato aminoacylated tRNA è protetto dall'ossidazione e uncharged tRNA non è. Periodato dopo trattamento di ossidazione della ricarica del tRNA viene utilizzata per stimare i livelli di carica del RNA raccolto. Tuttavia, la ricarica dei diversi tRNAs ha dimostrata di essere colpiti dal trattamento fornendo così qualche inesattezza12. Il metodo qui presentato misure carico direttamente da RNA purificato, escludendo quindi eventuali pregiudizi da reazioni chimiche o enzimatiche. Una limitazione di questo metodo è che solo una specie di tRNA viene rilevato in un momento, così anche se la stessa Northern blot può essere spogliato e reprobed per più tRNAs, è che richiede tempo e un po ' laborioso per raccogliere dati su molti tRNAs.
Un modo affidabile di normalizzazione dei campioni a vicenda è fondamentale negli studi dove l'obiettivo è quello di confrontare i livelli relativi di una molecola attraverso diversi campioni. Qui introduciamo una procedura di normalizzazione per RNA dove una piccola aliquota di Escherichia coli cellule che overexpressing il tRNA raraselC è aggiunto come spike-in per tutti i campioni sperimentali prima Purificazione RNA. Questo metodo è utile non solo quando si esaminano i tRNAs ma per quantificazione relativa di ogni specie di RNA quando la messa a punto sperimentale è tale che nessun RNA endogeno può essere attendibile per essere presenti alla stessa concentrazione cellulare in tutti i campioni. Ad esempio, il livello di una "pulizia" RNA ribosomiale RNA-come spesso viene utilizzato come un riferimento endogeno per confrontare le relative quantità di RNA un altro tra i diversi campioni13. Ma questo è di scarsa utilità se la concentrazione cellulare del RNA riferimento varia tra i campioni, come può essere il caso per il RNA ribosomiale (rRNA) se il campionamento avviene durante un sforzo risposta15,16,17 o durante entrata in fase stazionaria17. L'aggiunta di spike-in cellule di campioni sperimentali prima Purificazione RNA fornisce un modo solido e preciso di normalizzazione indipendenti del setup di campionamento. Un altro modo di standardizzazione è l'aggiunta di uno o più RNA di spike ai campioni sperimentali dopo la purificazione di RNA. Tuttavia, questo metodo non tiene conto delle eventuali differenze di recupero di RNA tra campioni.
Utilizzando cellule di Escherichia coli che iperesprimere il riferimento RNA come spike-in celle (Vedi Figura 1) in un esperimento su Escherichia coli ha lo svantaggio che risulta inoltre di una piccola quantità di esogeno Escherichia coli totale RNA per la campioni. Correggiamo per questa aggiunta analizzando un campione contenente solo spike-in celle in parallelo con i campioni sperimentali (Vedi il Northern blot nel Figura 2, lane etichettato "selC"). Il protocollo presentato è stato sviluppato per e. coli K-12 ma è probabile che sia applicabile per la maggior parte delle specie batterica.

<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

RNA transfer (tRNA) è una parte essenziale dell'apparato traduzionale in qualsiasi organismo. tRNAs legano e trasferire gli aminoacidi al ribosoma traduzione. I livelli relativi di tRNAs differenti ed il rapporto di aminoacylated tRNA per tRNA totale, conosciuto come il livello di carico, sono fattori importanti nel determinare l'accuratezza e la velocità di traduzione. Di conseguenza, l'abbondanza e livelli di carica di tRNAs sono variabili importanti per misurare quando si studia la sintesi delle proteine, ad esempio in varie condizioni di stress. Qui, descriviamo un metodo per la raccolta del tRNA e misurando direttamente l'abbondanza relativa e il livello di carico assoluto di determinate specie di tRNA in Escherichia coli. Il tRNA è raccolto in modo tale che il legame labile tra il tRNA e l'aminoacido è conservato. il RNA è quindi sottoposto a elettroforesi su gel e Northern blotting, che si traduce nella separazione dei tRNAs cariche e scariche. I livelli di specifici tRNAs nei diversi campioni possono essere paragonati a causa dell'aggiunta di spike-a celle per la normalizzazione. Prima Purificazione RNA, aggiungiamo 5% delle cellule di Escherichia coli che overproduce la rara di tRNAselC a ciascun campione. La quantità di specie di interesse in un campione di tRNA è poi normalizzata alla quantità di tRNAselC nello stesso campione. Aggiunta di spike-in cellule prima Purificazione RNA ha il vantaggio rispetto alla aggiunta di purificato RNA spike-in che è anche responsabile di eventuali differenze di efficienza di lisi delle cellule fra i campioni.

Utilizzando la procedura descritta qui, l'abbondanza e ricarica i livelli di tre tRNAs sono stati misurati in e. coli K-12 prima e durante l'inedia dell'aminoacido.
Il auxotroph di arginina ceppo NF9154 (thr leu suo argH thi mtl supE44) è stato coltivato per almeno dieci generazioni in MOPS supplementato con 0,4% glicerolo, 50 µ g/mL treonina, leucina, arginina e istidina di 5 µ g/mL ...

Watch this Scientific Journal Video about Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli at JoVE.com

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

Quantificazione di abbondanza e ricarica i livelli di RNA di trasferimento in Escherichia coli

Qui presentiamo un metodo per misurare direttamente il RNA di trasferimento carica livelli da purificato Escherichia coli RNA così come un modo per confrontare i livelli relativi di RNA di trasferimento, o qualsiasi altri RNA breve, attraverso diversi campioni basati sull'aggiunta di spike-a cellule che esprimono un gene di riferimento.

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

Research

JoVE Journal

Biochemistry

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

9.2K Views.  University of Copenhagen. Qui presentiamo un metodo per misurare direttamente il RNA di trasferimento carica livelli da purificato Escherichia coli RNA così come un modo per confrontare i livelli relativi di RNA di trasferimento, o qualsiasi altri RNA breve, attraverso diversi campioni basati sull'aggiunta di spike-a cellule che esprimono un gene di riferimento.

Video: 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.

Analisi del rallentamento del gel agarosio proteina-tRNA per l&#39;analisi del<em&gt; N</em<sup&gt; 6</sup&gt; -reonilcarbamoyladenosine TcdA Funzione

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 ...

Ricerca

Biochimica

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.

DNA-RNA simultanea Estrazione da sedimenti costieri e la quantificazione dei geni 16S rRNA e trascrizioni da 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 ...

Ambiente

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.

In tempo reale analisi del fattore di trascrizione associazione, trascrizione, traduzione e fatturato per visualizzare eventi globali durante l'attivazione cellulare

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

Genetica

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.

Etichettatura metabolica e profilatura di RNA di trasferimento utilizzando 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 ...

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.

Un saggio per quantificare il legame proteino-RNA nei batteri

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

Analisi della dinamica del tRNA con AQRNA-seq

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.

Autore in primo piano: Ruolo di AQRNA-seq nella mappatura di piccoli RNA e nel disfacimento dei meccanismi di traduzione delle proteine

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.

AQRNA-seq per la quantificazione di piccoli RNA

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.

RIBO-seq in Bacteria: un protocollo di raccolta dei campioni e preparazione della biblioteca per il sequenziamento NGS

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.

Genoma Analisi dei Aminoacylation (carica) I livelli di tRNA usando i 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 ...

Quantificazione di abbondanza e ricarica i livelli di RNA di trasferimento in Escherichia coli

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Analisi del rallentamento del gel agarosio proteina-tRNA per l'analisi del

DNA-RNA simultanea Estrazione da sedimenti costieri e la quantificazione dei geni 16S rRNA e trascrizioni da Real-time PCR

In tempo reale analisi del fattore di trascrizione associazione, trascrizione, traduzione e fatturato per visualizzare eventi globali durante l'attivazione cellulare

Etichettatura metabolica e profilatura di RNA di trasferimento utilizzando Macroarrays

Un saggio per quantificare il legame proteino-RNA nei batteri

AQRNA-seq per la quantificazione di piccoli RNA

AQRNA-seq per la quantificazione di piccoli RNA

AQRNA-seq per la quantificazione di piccoli RNA

RIBO-seq in Bacteria: un protocollo di raccolta dei campioni e preparazione della biblioteca per il sequenziamento NGS

Genoma Analisi dei Aminoacylation (carica) I livelli di tRNA usando i microarrays