tRNAs Are Stable After All: Pitfalls in Quantification of tRNA from Starved Escherichia coli Cultures Exposed by Validation of RNA Purification Methods
Thomas Prossliner, Shreya Agrawal, Ditte F. Heidemann, Michael A. Sørensen, Sine Lo Svenningsen
Abstract
We show that E. coli tRNAs are remarkably stable during several days of nutrient starvation, although rRNA is degraded extensively under these conditions. The levels of these two major RNA classes are considered to be strongly coregulated at the level of transcription. We demonstrate that E. coli can control the ratio of tRNAs per ribosome under starvation by means of differential degradation rates. The question of tRNA stability in stressed E. coli cells has become subject to debate. Our in-depth analysis of RNA quantification methods reveals hidden technical pitfalls at every step of the analysis, from RNA extraction to target detection and normalization. Most importantly, starved E. coli populations were more resilient to RNA extraction than unstarved populations. The current results underscore that the seemingly trivial task of quantifying an abundant RNA species is not straightforward for cells cultured outside the exponential growth regime.