Name: quantification of the abundance and charging levels of transfer rnas in escherichia coli
Uploaded: 2017-08-22T14:00:00
Description: Watch this Scientific Journal Video about Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli at JoVE.com

The authors thank Marit Warrer for excellent technical assistance. This work was supported by the Danish Council for Independent Research | Natural Sciences [1323-00343B] and the Danish National Research Foundation [DNRF120].

1. Preparation of bacterial cultures.
<ol>
	<li>Grow all cultures in Erlenmeyer flasks with a culture/flask volume ratio of at most 1/6. In a typical experimental setup, grow the cultures at 37.0 &#176;C and shaking at 160 rpm in morpholinepropanesulfonic acid (MOPS) minimal medium18 supplemented with the preferred carbon source, for example 0.2% glucose, for at least 10 consecutive generations before RNA harvest.</li>
	<li>The day before RNA harvest, dilute an outgrown culture of the desired st...

<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. 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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>

This protocol describes how to simultaneously measure the charging level of specific E. coli tRNAs and compare the relative levels of the tRNAs in different samples. The critical points of the protocol are 1) to handle the samples in such a way that the cellular tRNA charging levels are retained, 2) to normalize tRNA quantities in such a way that relative tRNA levels in different samples can be reliably compared, and 3) to ensure the specificity of the selected probes for the tRNAs of interest. These points are ...

In the following, we present a method for quantifying specific tRNAs and measuring their charging levels by Northern blotting. The method is based on a technique first developed by Varshney et al.1. By harvesting cells into trichloroacetic acid (TCA) and keeping the samples at 0 &#176;C throughout the RNA purification, the ester bond between the tRNA and the amino acid is conserved2,3,4. Aminoacylated tRNAs can be distinguished from their nonacylated counterparts by gel electrophoresis and Northern blotting, due to a decreased mobility of the aminoacylated tRNA in the gel, caused by the covalently bound amino acid5. Additionally, we present a protocol for normalizing tRNA quantities by addition of spike-in cells overexpressing the rarely used tRNAselC 4.
tRNAs are some of the most abundant molecules in the bacterial cell and an absolutely vital part of the translation machinery. tRNAs bind amino acids and transfer them to the translating ribosomes. The binding of amino acids to tRNAs (aminoacylation or charging) is facilitated by aminoacyl tRNA synthetases. The relative abundance of different charged tRNAs is important for ensuring the fidelity of protein synthesis, because underrepresentation of the cognate charged tRNA for a given messenger RNA (mRNA) codon increases the likelihood that a near-cognate tRNA will erroneously deliver its amino acid to the growing polypeptide chain on the ribosome6. The importance of charged tRNA is reflected in the extensive response of the E. coli cell to a severe drop in the charging levels of a tRNA; the stringent response. During the stringent response the synthesis of tRNAs, ribosomal RNAs and most mRNAs is lowered in favor of transcription of specific mRNAs associated with amino acid biosynthesis and stress survival, and the growth rate of the cells is lowered dramatically7. Furthermore, recent work by us and others has shown that E. coli actively degrades the majority of its tRNA in response to stresses that limit translation4,8, suggesting that adjustment of the tRNA levels may be important for coping with such stresses. Thus, reliable measurements of tRNA abundances and charging levels will be an important tool for fully understanding bacterial stress responses.
E.coli is commonly used to express recombinant protein and due to the differences in codon usage between species, suboptimal expression is a problem often faced9. This can be circumvented by expression of additional tRNAs needed to translate the recombinant mRNA10. Measurements of tRNA charging levels in such strains could guide troubleshooting efforts and help optimize protein expression.
This method also enables the detection of &#34;mischarging&#34;; a tRNA molecule aminoacylated with a non-cognate amino acid. Aminoacylation by different amino acids may cause a tRNA to migrate with slightly different velocities through polyacrylamide gels1,11. In some cases, the method can also be used to distinguish different modification patterns on otherwise identical tRNAs3.
Another established biochemical procedure for the investigation of tRNA charging levels is periodate oxidation. The method relies on the observation that aminoacylated tRNA is protected from periodate oxidation and uncharged tRNA is not. After periodate oxidation treatment the recharging of the tRNA is used to estimate the charging levels of the harvested RNA. However, the recharging of several tRNAs has been shown to be affected by the treatment thus providing some inaccuracy12. The method presented here measures charging directly from purified RNA, thus excluding any biases from chemical or enzymatic reactions. One limitation of this method is that only one tRNA species is detected at a time, so although the same Northern blot can be stripped and reprobed for multiple tRNAs, it is time-consuming and somewhat laborious to collect data on many tRNAs.
A reliable way of normalizing samples to each other is vital in studies where the goal is to compare the relative levels of a molecule across different samples. Here we introduce a normalization procedure for RNA where a small aliquot of E. coli cells overexpressing the rare tRNAselC is added as a spike-in to all the experimental samples prior to RNA purification. This method is useful not only when examining tRNAs but for relative quantification of any species of RNA when the experimental setup is such that no endogenous RNA can be trusted to be present at the same cellular concentration in all the samples. For example, the level of a &#34;housekeeping&#34; RNA-like ribosomal RNA is often used as an endogenous reference to compare the relative quantities of another RNA between different samples13. But this is of little use if the cellular concentration of the reference RNA varies between samples, as can be the case for ribosomal RNA if sampling occurs during a stress response15,16,17 or during entry into stationary phase17. The addition of spike-in cells to the experimental samples prior to RNA purification provides a solid and accurate way of normalization independent of the sampling setup. Another way of standardization is the addition of one or more spike-in RNAs to the experimental samples after RNA purification. However, this method does not account for any differences in RNA recovery between samples.
Using E. coli cells that overexpress the reference RNA as spike-in cells (see Figure 1) in an experiment on E. coli has the drawback that it results in addition of a small amount of exogenous E. coli total RNA to the samples. We correct for this addition by analyzing a sample containing only spike-in cells in parallel with the experimental samples (see the Northern blot in Figure 2, lane labeled &#34;selC&#34;). The protocol presented is developed for E. coli K-12 but is likely to be applicable for most bacterial species.

<table>
	<tbody>
		<tr>
			<td>Urea</td>
			<td>Merck</td>
			<td>57-13-6</td>
			<td>Purity &#62;99%</td>
		</tr>
		<tr>
			<td>Polyacrylamide</td>
			<td>Serva</td>
			<td>79-06-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Bis (N,N&#39;-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 &#947;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+ 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&#252;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</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

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Using the procedure described here, the abundance and charging levels of three tRNAs were measured in E. coli K-12 before and during amino acid starvation.
The arginine auxotroph strain NF9154 (thr leu his argH thi mtl supE44) was grown for at least ten generations in MOPS minimal medium supplemented with 0.4% glycerol, 50 &#181;g/mL threonine, leucine, arginine, and 5 &#181;g/mL histidi...

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

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

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

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

The overall goal of this experiment is to quantify the abundance and charging levels of transfer RNAs in Escherichia coli. This method can help answer key questions in cell biology such as, how tRNA charging levels changes at different growth states. The main advantage of this method is that, RNA levels can be quantitatively compared even between growth states that drastically alter the composition of the RNA in the cells.Start this experiment by growing experimental and spike-in E.coli cultures according to the text protocol. Grow the spike-in culture at 37 degree celsius, while shaking at 160 RPM. When the spike-in culture has reached an OD436 of about 0.1, induce the expression of tRNA cell C by adding IPTG.Grow the culture until it reaches an OD436 of about 0.5. Then, move the culture to ice until all samples have been harvested. After that, prewarm one 100 milliliter capped culture flask containing four milliliters of 10%TCA for each sample in a 37 degree celsius water bath.Measure and record the OD436 of the experimental cultures directly before cell harvest. Then, transfer four milliliters of culture to a prewarmed TCA containing flask. Mix the sample by hand swirling the flask for five seconds, which will immediately disable all enzymatic activities preserving the charging levels of the tRNAs.When all samples have been collected, add 5%spike-in cells to each sample. Also prepare a control sample consisting only of spike-in cells as described in the text protocol. To start RNA extraction, pour eight milliliters of each sample, including controls into an ice chilled centrifuge tube.Centrifuge the samples at 9, 500 x G at four degree celsius for 10 minutes. After removing the supernatant completely, re-suspend the pellet in 0.3 milliliters of cold sodium acetate, containing 10 millilimolar EDTA. Transfer each samples into a cold 1.5 milliliter microcentrifuge tube and add 0.3 milliliters of phenol equilibrated with the buffer, used to re-suspend the pellet.Then, vortex each sample 10 times for 15 seconds each time. With at least one minute at zero degree celsius between each vortex, to keep the samples cold. Next, centrifuge the samples at 20, 000 x G at four degree celsius for 15 minutes.Transfer the upper or water face to a new 1.5 milliliter microcentrifuge tube. Add 0.3 milliliters of cold phenol to the water face and vortex the samples four times 15 seconds each as previously described. Then, centrifuge the samples at 20, 000 x G at four degrees celsius for 10 minutes.Transfer the water face to a new cold 1.5 milliliter microcentrifuge tube and add 2.5 volumes of cold 96%ethanol. Then, incubate the tubes at 20 degree celsius for one hour to precipitate the RNA. Centrifuge the samples at 20, 000 x G at four degree celsius for 30 minutes.After removing the supernatant, carefully add one milliliter of cold 70%ethanol to the RNA pellet, making sure not to disturb it. Invert the tube gently once, to wash it with ethanol. Finally, centrifuge at 20, 000 x G at four degree celsius for 10 minutes.Remove the supernatant completely, and air dry the pellet until the ethanol is completely removed. At 20 microliters of cold sodium acetate containing 1 millimolar EDTA, vortex vigorously to re-suspend the pellet. To start electrophoresis, mix four microliters of each sample with six microliters of loading buffer.Load the samples including controls onto a 0.4 millimeter thick 6.5 polyacrylamide gel containing eight molar ureal and 0.1 molar sodium acetate buffer. Then, separate the RNA by running the electrophoresis at 10 volts per centimeter of gel at four degree celsius for approximately 20 hours until the bromophenol blue dye reaches the bottom of the gel. Carefully separate the two glass plates so that the gel remains on one of them.Use the gel area from the xylene cyanol dye and 20 centimeters down towards the bromophenol dye for blotting. Cut a thin piece of filter paper to the size of the desired blotting area. Place the filter paper on top of the part of the gel that will be used for blotting.Cut off and discard the parts of the gel not covered with filter paper. Then use the filter paper to carefully lift the gel away from the glass plate. Place a positively charged nylon membrane on top of the gel, and electroblot it in transfer buffer at 20 volts for 90 minutes.Finish by crosslinking the RNA to the membrane using UV light. Next, place the crosslinked membrane in a hybridization tube and add six milliliters of hybridization solution. Rotate the tube at 42 degree celsius for one hour to pre-hybridize the membrane.Then, add 30 peak molar radioactive oligo DNA probe, five prime and labeled with P32. Rotate to incubate the membrane with the probe at 42 degree celsius overnight. After incubation, use a disposable 10 milliliter pipette to remove the probe.Next, quickly wash the membrane twice in the hybridization tube, in 15 milliliters of washing solution at room temperature. Move the membrane to a flat plastic container, cover it with the washing solution and close the lid. Incubate the membrane on a shaker at room temperature for 30 minutes.Change the solution and continue washing until achieving a satisfying signal to noise ratio. Use a geiger muller tube to obtain the signal to noise ratio, by comparing the radiation from the area where the probe has hybridized to tRNA, to the radiation from an empty area. After that, wrap the membrane tightly in plastic wrap and seal it airtight by welding.Place this membrane on a phosphorimaging screen. After adequate exposure time, scan the screen using a laser scanner and save the file in gel format. Next, load the gel file into the imaging software.Draw a vertical line along each of the sample lanes so that it spans most of the width of the lane while excluding the edges of the bands where the signal can be uneven. To visualize counts from each lane, click analysis and then, create graph. Manually define the two peaks that represent the charged and the uncharged tRNA from each lane.After that, click on analysis and then, area report to obtain counts from each peak. Finally, record the area under each of the two peaks. Calculate the tRNA levels as described in the text protocol.This method was used to measure the abundance of various tRNAs and E.Coli nf915 before and during our arginine starvation. On the northern plot hybridized with different tRNA probes, there is a clear separation between the bands that represent the charge tRNA from the bands that represent the uncharged tRNA. The radiation profile along each lane, was obtained from the northern blots using imaging software.The radiation signal from each lane was quantified by isolating each from the blot. A graph that represents the quantification of the signal from a single lane, shows two peaks. The left peak comes from the aminoacylated tRNA and the right from the deacylated tRNA.The area under each peak is exported for further analysis. Quantification of the bands from the northern blot, shows that the relative abundance of all tested tRNA species rapidly and significantly decreased during amino acid starvation. On the other hand, tRNA charging levels of the arginine accepting tRNA dropped rapidly upon arginine removal and then slightly increased during the starvation period.However, the charging levels of the other tRNAs increased slightly upon starvation and then decreased to almost pre-starvation levels. After watching this video, you should have a good understanding of how to purify RNA in a way that tRNA charging and abundance can be quantified and how to use spike-in cells for normalization. Throughout this procedure, it's important to remember to keep the RNA cold and acidic to prevent the hydrolysis of the RNA.

Research

JoVE Journal

Biochemistry

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

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