The Role of the Codon and the Initiation Factor IF‐2 in the Selection of N‐Blocked Aminoacyl‐tRNA for Initiation

Laken, K van der; Bakker-Steeneveld, H; Berkhout, Ben; Knippenberg, P.H. Van

doi:10.1111/j.1432-1033.1980.tb04394.x

Cited by 11 publications

(2 citation statements)

References 14 publications

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“…On the contrary, mRNAs with a UUC codon at the start site showed negligible thermophoresis signal when Bpy-tRNAi was used, indicating that MST monitors the formation of the 30S IC rather than the 30S pre-IC. To further validate this observation, we used a Bpy-Phe-tRNA Phe with the corresponding UUC codon at the start site, which resulted in an increase of thermophoresis, consistent with IF2 requiring an N-blocked aminoacyl tRNA for translation initiation [26,27]. Accordingly, if an AUG codon was used at the start site, negligible MST amplitude was observed ( Fig 1D).…”

Section: Experimental Approachmentioning

confidence: 59%

How the initiating ribosome copes with ppGpp to translate mRNAs

et al. 2020

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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.

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Section: Experimental Approachmentioning

confidence: 59%

How the initiating ribosome copes with ppGpp to translate mRNAs

et al. 2020

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“…If acetylated, the amine group of the A site amino acid would be unavailable to the carboxyl group and protein synthesis would be blocked ( Figure 5 A). This is the basis for translation arrest by chemical acetylation of aminoacyl-tRNA ( Cuzin et al., 1967 , van der Laken et al., 1980 ).…”

Section: Resultsmentioning

confidence: 99%

A Salmonella Toxin Promotes Persister Formation through Acetylation of tRNA

et al. 2016

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SummaryThe recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.

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