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

Summary

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.

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

Introduction

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 °C throughout the RNA purification, the ester bond between the tRNA and the amino acid is conserved^2,3,4. Aminoacylated tRNAs can be distinguished from their nonacylated counterparts by gel electrophoresis and Northern blotting, due to a decreased mobility of the ....

Protocol

1. Preparation of bacterial cultures.

1. 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 °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.
2. The day before RNA harvest, dilute an outgrown culture of the desired strain in MOPS minimal medium in such a way that it will reach the desired OD₄₃₆ at the desired time the following day. Calculate the volume of ou....

Results

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 µg/mL threonine, leucine, arginine, and 5 µg/mL histidine at 37 °.......

Discussion

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 .......

Materials

List of materials used in this article

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