Quantification of the Abundance and Charging Levels of Transfer RNAs in <em>Escherichia coli</em>

Abstract

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 tRNA to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNA 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.

Short‐term kinetics of rRNA degradation in Escherichia coli upon starvation for carbon, amino acid or phosphate

“…Here, we used a method relying on the addition of spike‐in cells expressing high amounts of a reference RNA to accurately quantify bacterial RNA content. The advantage of the spike‐in method is that it allows for normalization of sample signals, which is required to reduce noise caused by variations in RNA recovery and blotting, without making assumptions about the existence of an invariable endogenous reference RNA or invariable total RNA contents of the cells, before and after starvation (Sorensen, Fehler, & Svenningsen, ; Stenum, Sorensen, & Svenningsen, ; Svenningsen et al, ). Although RNA was extracted with hot phenol (see Experimental Procedures), which efficiently disrupts gram‐negative bacterial membranes (Ares, ), recovery of RNA from starved cells could be reduced compared to growing cells due to physiological changes of the cell envelope and size of the cells.…”

Transfer RNA instability as a stress response inEscherichia coli: Rapid dynamics of the tRNA pool as a function of demand

“…45,46 To assess whether PAP I is necessary for the general tRNA decay in the early response to amino acid starvation, we measured tRNA levels in a rph + derivative of MG1655 and the isogenic ΔpcnB strain, using the method described previously. 14,21 Fig. 1 shows that all measured tRNA half-lives during starvation for isoleucine were unaffected by the absence of PAP I.…”

“…14,21 By quantifying a reference RNA that is highly expressed in the spike-in cells and either absent or present at very low levels in the experimental samples, it is possible to obtain accurate normalization by adding only a small aliquot of spike-in cells to the experimental samples. We typically add 1–5% spike-in cells to each sample (based on optical density), and load a control sample containing only spike-in cells to quantify the contribution of the spike-in cells to the signal from the RNA of interest.…”

“…We typically add 1–5% spike-in cells to each sample (based on optical density), and load a control sample containing only spike-in cells to quantify the contribution of the spike-in cells to the signal from the RNA of interest. 21 The spike-in-cell approach has the clear advantage that no assumptions about constancy of the levels of the reference RNA across the tested conditions are necessary, because the spike-in cells are added after harvest of the experimental samples. Addition of the spike-in cells prior to RNA extraction (as opposed to the addition of purified spike-in transcripts after RNA purification) ensures that the reference RNA also reflects any sample-to-sample variation in RNA recovery.…”

“…Transfer RNA counts quantified from a northern blot as described in ref. 14, 21. In brief, cultures of MG1655 rph + ΔpcnB::cat-sacB (PcnB) and MG1655 rph + (WT) were grown in MOPS minimal media 98 supplemented with 0.2% glucose for at least ten generations in exponential phase prior to isoleucine starvation, which was induced by the addition of 400 μg/ml valine.…”