Transfer RNA is highly unstable during early amino acid starvation inEscherichia coli

Abstract

Due to its long half-life compared to messenger RNA, bacterial transfer RNA is known as stable RNA. Here, we show that tRNAs become highly unstable as part of Escherichia coli's response to amino acid starvation. Degradation of the majority of cellular tRNA occurs within twenty minutes of the onset of starvation for each of several amino acids. Both the non-cognate and cognate tRNA for the amino acid that the cell is starving for are degraded, and both charged and uncharged tRNA species are affected. The alarmone ppGpp orchestrates the stringent response to amino acid starvation. However, tRNA degradation occurs in a ppGpp-independent manner, as it occurs with similar kinetics in a relaxed mutant. Further, we also observe rapid tRNA degradation in response to rifampicin treatment, which does not induce the stringent response. We propose a unifying model for these observations, in which the surplus tRNA is degraded whenever the demand for protein synthesis is reduced. Thus, the tRNA pool is a highly regulated, dynamic entity. We propose that degradation of surplus tRNA could function to reduce mistranslation in the stressed cell, because it would reduce competition between cognate and near-cognate charged tRNAs at the ribosomal A-site.

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

“…This finding, together with the finding that the kinetics of tRNA decay upon amino acid starvation are similar in wild-type E. coli and a relA mutant, suggests that tRNA instability is not a unique consequence of the stringent response, but may occur as a general response to stresses that reduce translation. 14 The argument is supported by a recent report of extensive tRNA degradation during oxidative stress, 30 which causes stalling of ribosomes at 8-oxoG residues in the mRNA 31 and thereby also limits the pool of functionally intact mRNA available for translation. Since the capacity to synthesize new protein is severely reduced during amino acid starvation, a tRNA-degradation mechanism that depends on synthesis of new protein may not be feasible.…”

“…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.…”

“…Using the spike-in-cell approach, we showed that the majority of cellular tRNA is degraded within twenty minutes of the onset of a sudden amino acid starvation. 14 …”

“…Consistently, we found that global tRNA degradation occurs even in cultures that are pre-treated with chloramphenicol, revealing that a large capacity for tRNA degradation exists, which does not require de novo protein synthesis. 14 The simplest model to explain these observations is that there is no molecular signal per se that triggers tRNA degradation. Instead, we propose a demand-based model for tRNA degradation, in which tRNAs are protected from degradation when they are occupied by binding to members of the translational machinery (the aminoacyl-tRNA-synthetases, elongation factor Tu, and the ribosomes), but they become accessible for degradation once they are not engaged in charging and translation.…”

“…This observation gave rise to a working model, in which a tRNA becomes substrate for degradation whenever the tRNA supply exceeds the demand for tRNAs in protein synthesis. 14 We begin this point-of-view article by underlining the importance of careful normalization of gene expression data obtained from stress conditions that disrupt steady-state growth, which may explain why tRNA degradation has not been described earlier.…”