How the initiating ribosome copes with ppGpp to translate mRNAs

Abstract

During host colonization, bacteria use the alarmones (p)ppGpp to reshape their proteome by acting pleiotropically on DNA, RNA, and protein synthesis. Here, we elucidate how the initiating ribosome senses the cellular pool of guanosine nucleotides and regulates the progression towards protein synthesis. Our results show that the affinity of guanosine triphosphate (GTP) and the inhibitory concentration of ppGpp for the 30S-bound initiation factor IF2 vary depending on the programmed mRNA. The TufA mRNA enhanced GTP affinity for 30S complexes, resulting in improved ppGpp tolerance and allowing efficient protein synthesis. Conversely, the InfA mRNA allowed ppGpp to compete with GTP for IF2, thus stalling 30S complexes. Structural modeling and biochemical analysis of the TufA mRNA unveiled a structured enhancer of translation initiation (SETI) composed of two consecutive hairpins proximal to the translation initiation region (TIR) that largely account for ppGpp tolerance under physiological concentrations of guanosine nucleotides. Furthermore, our results show that the mechanism enhancing ppGpp tolerance is not restricted to the TufA mRNA, as similar ppGpp tolerance was found for the SETI-containing Rnr mRNA. Finally, we show that IF2 can use pppGpp to promote the formation of 30S initiation complexes (ICs), albeit requiring higher factor concentration and resulting in slower transitions to translation elongation. Altogether, our data unveil a novel regulatory mechanism at the onset of protein synthesis that tolerates physiological concentrations of ppGpp and that bacteria can exploit to modulate their proteome as a function of the nutritional shift happening during stringent response and infection.

The alarmones (p)ppGpp are part of the heat shock response of Bacillus subtilis

“…A set of interesting in vitro experiments suggested a modulating effect of ppGpp and (p)ppGpp on IF2 and translation initiation, which also depended on specific structured elements of translated mRNA, allowing translation of specific mRNA's in the presence of alarmones [41]. These experiments indicate a specific ability of alarmones, interacting with IF2, to limit translation in general, while possibly still allowing the expression of specific genes necessary for stress resistance [41].…”

“…In addition to this protective function, (p)ppGpp, which is synthesized only as a pulse by Rel during relatively uninhibited growth at 50˚C, could modulate or inhibit translation, presumably by directly interfering with translation factors [38,39,41,69]. A set of interesting in vitro experiments suggested a modulating effect of ppGpp and (p)ppGpp on IF2 and translation initiation, which also depended on specific structured elements of translated mRNA, allowing translation of specific mRNA's in the presence of alarmones [41]. These experiments indicate a specific ability of alarmones, interacting with IF2, to limit translation in general, while possibly still allowing the expression of specific genes necessary for stress resistance [41].…”

“…In addition, the global regulator and repressor CodY is activated by GTP via an allosteric binding site, and therefore amino acid biosynthesis genes and other pathways are de-repressed upon a drop of the cellular GTP level during the SR [36,37]. Beyond its impact on transcription, (p)ppGpp can modulate ribosome assembly, translation initiation and elongation by binding, for example, to the translation initiation factor IF-2 and other ribosome-associated GTPases [38][39][40][41][42][43][44]. With its ability to inhibit translation and growth, the SR was also implicated in persister cell formation and development of antibiotic tolerance [45].…”

Possible Roles for Basal Levels of (p)ppGpp: Growth Efficiency Vs. Surviving Stress

“…(p)ppGpp can bind to several GTPases involved in translation or ribosome biogenesis, including IF2, inhibiting their activity ( Zhang et al, 2018 ).The end result would be a decrease on the number of active ribosomes, now susceptible of degradation. Interestingly, the inhibitory effects of (p)ppGpp over IF2 activity may depend on the mRNA being translated ( Vinogradovaid et al, 2020 ).…”

(p)ppGpp inhibits 70S ribosome formation inStaphylococcus aureusby impeding GTPase-ribosome interactions

“…Ribosomal rRNA production and biogenesis are not the only aspects of protein synthesis that (p)ppGpp regulates, given its ability to bind to the bacterial initiation factor 2 (IF2), elongation factor Tu (EF-Tu), elongation factor G (EFG), elongation factor Ts (EFTs) and release factor 3 (RF3) (58)(59)(60)(61)(62). In each case, competitive binding of (p)ppGpp to these GTPases results in an inhibition of activity and reduction of the elongation cycle.…”