How the initiating ribosome copes with ppGpp to translate mRNAs

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.Russian Foundation for Basic ResearchRevisión por pare

Abstract P-29: Cryoem Study of the Inhibition of Bacterial Ribosomes by Madumycin II

Background: The efficiency of widely used antibiotics is limited by continuous improvement of resistance mechanisms. Thus, the research of poorly studied drugs that have not received practical use until now becomes relevant again. Protein translation is one of the major targets for antibiotics. Madumycin II (MADU) is an antibiotic of the streptogramin A class that binds to the peptidyl transferase center of the initiated bacterial 70S ribosome inhibiting the first cycle of peptide bond formation (I.A. Osterman et al. Nucleic Acids Res., 2017). The ability of MADU to interfere with translating ribosome is an open question that we address by investigation of high-resolution cryo-EM structures of MADU bound 70S ribosome complexes from Escherichia coli. Methods: Purified initiated and translating ribosome complexes preincubated with MADU were applied onto freshly glow discharged carbon-coated grids (Quantifoil R 1.2/1.3) and flash-frozen in the liquid ethane pre-cooled by liquid nitrogen in the Vitrobot Mark IV. Frozen grids were transferred into an in-house Titan Krios microscope. Data were collected using EPU software. Movie stacks were preprocessed in Warp software. For image processing, we have used several software packages: Relion 3.1, CryoSPARC, and CisTEM. The model was built in Coot. Results: We have obtained high-resolution cryo-EM structures of two ribosomal complexes with MADU before and after the first cycle of peptide bond formation with an average resolution of 2.3 Å. Preliminary analysis of the structures shows no major differences in the MADU binding mode to the ribosomal complexes under study suggesting that the quantity of amino acid residues attached to the P-site tRNA does not impact MADU bonding. Moreover, in both cases, we observed similar destabilization of the CCA-ends of A- and P-site tRNAs underlining the comparable influence of MADU on the ribosomal complexes. Conclusion: Our results suggest that although MADU binding site is located in the peptidyl transferase center, the presence of the second amino acid residue on the P-site tRNA does not preclude antibiotic binding. We assume that further elongation of the polypeptide chain would not have any impact either. High conformational lability of the CCA-ends of tRNA at the A and P sites upon binding of MADU obviously plays an important role in the inhibition mechanism of the bacterial ribosome. The further structural and biochemical analysis will be necessary to shed more light on the detailed mechanism of MADU action

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.Russian Foundation for Basic ResearchRevisión por pare

Biodiversity, drug discovery, and the future of global health:Introducing the biodiversity to biomedicine consortium, a call to action

First paragraph: Looking to nature for medicine is nothing new – we have been doing it for tens of thousands of years and although modern pharmaceutical science has come a long way from those ancient roots, nature is and will always be an important source of useful compounds and inspiration. Dismissing nature in this regard is a huge mistake as evolution is the greatest problem solver and the myriad compounds produced by the immense variety of species we share the planet with have been honed by three billion years of trial and error. However, with every bit of habitat that disappears under the plough or concrete we impoverish nature and deprive ourselves of potential medicines.Additional co-authors: Uttam Babu Shrestha, Milica Pešić, Alexander Kagansk

The structure of helix 89 of 23S rRNA is important for peptidyl transferase function of Escherichia coli ribosome

AbstractHelix 89 of the 23S rRNA connects ribosomal peptidyltransferase center and elongation factor binding site. Secondary structure of helix 89 determined by X-ray structural analysis involves less base pairs then could be drawn for the helix of the same primary structure. It can be that alternative secondary structure might be realized at some stage of translation. Here by means of site-directed mutagenesis we stabilized either the “X-ray” structure or the structure with largest number of paired nucleotides. Mutation UU2492-3C which aimed to provide maximal pairing of the helix 89 of the 23S rRNA was lethal. Mutant ribosomes were unable to catalyze peptide transfer independently either with aminoacyl-tRNA or puromycin

Biodiversity, drug discovery, and the future of global health: Introducing the biodiversity to biomedicine consortium, a call to action

Looking to nature for medicine is nothing new – we have been doing it for tens of thousands of years and although modern pharmaceutical science has come a long way from those ancient roots, nature is and will always be an important source of useful compounds and inspiration. Dismissing nature in this regard is a huge mistake as evolution is the greatest problem solver and the myriad compounds produced by the immense variety of species we share the planet with have been honed by three billion years of trial and error. However, with every bit of habitat that disappears under the plough or concrete we impoverish nature and deprive ourselves of potential medicines