tRNAs Are Stable After All: Pitfalls in Quantification of tRNA from Starved Escherichia coli Cultures Exposed by Validation of RNA Purification Methods

Abstract

tRNAs and ribosomal RNAs are often considered stable RNAs. In contrast to this view, we recently proposed that tRNAs are degraded during amino acid starvation and drug-induced transcription inhibition. However, reevaluation of our experimental approach revealed that common RNA extraction methods suffer from alarming extraction and size biases that can lead to gross underestimation of RNA levels in starved Escherichia coli populations. Quantification of tRNAs suffers additional biases due to differing fractions of tRNAs with base modifications in growing versus starved bacteria. Applying an improved methodology, we measured tRNA levels after starvation for amino acids, glucose, phosphate, or ammonium and transcription inhibition by rifampicin. We report that tRNA levels remain largely unaffected in all tested conditions, including several days of starvation. This confirms that tRNAs are remarkably stable RNAs and serves as a cautionary tale about quantification of RNA from cells cultured outside the steady-state growth regime. rRNA, conversely, is extensively degraded during starvation. Thus, E. coli downregulates the translation machinery in response to starvation by reducing the ribosome pool through rRNA degradation, while a high concentration of tRNAs available to supply amino acids to the remaining ribosomes is maintained.