How the initiating ribosome copes with ppGpp to translate mRNAs.

Daria S Vinogradova,Elena Maksimova,Jose Alberto Nakamoto,Pavel Kasatsky,Andrey L Konevega,Alena Paleskava,Victor Zegarra,Pohl Milón,Wendy V Gilbert

doi:10.1371/journal.pbio.3000593

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.

Highlights

During colonization, pathogenic bacteria reshapes its transcriptome and proteome to activate virulence genes, promote tissue-associated biofilm and enter dormancy, increasing aggressiveness, antibiotic tolerance, and persistence of the pathogen
Addition of GTP increased the amplitude of thermophoresis stoichiometrically with Initiation Factor 2 (IF2), while in the presence of GDP, ppGpp or without any nucleotide (Apo), the amplitude of thermophoresis was lower (Fig. 2a), in agreement with IF2 requiring GTP for rapid recruitment of tRNAi to the 30S complex 19
GTP seems to activate IF2 to promote tRNAi binding to the 30S IC, 87 whereas, GDP and ppGpp prevent IF2 from recruiting the initiator tRNA

Summary

Introduction

Pathogenic bacteria reshapes its transcriptome and proteome to activate virulence genes, promote tissue-associated biofilm and enter dormancy, increasing aggressiveness, antibiotic tolerance, and persistence of the pathogen (reviewed in 1,2). Host colonization entails fluctuations of nutrient availability that, generally, triggers stringent response in bacteria (Fig. 1). Stringent response is mediated by the ribosome-associated RelA/SpoT homolog protein superfamily and triggers the accumulation of the hyperphosphorylated guanosine nucleotides, altogether called (p)ppGpp (Fig. 1) 3. Impaired (p)ppGpp production results in antibiotic sensitivity, low biofilm formation, and low pathogenicity of several bacteria, making the stringent response an appealing target for antibiotic development 4-6. MRNA translation to proteins requires the action of several guanosine nucleotide-binding factors, on and off the ribosome. (p)ppGpp have been shown to bind translational GTPases 7,8, the extent of inhibition and subsequent effect on protein synthesis regulation remain controversial

Methods

Results

Conclusion