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.

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation.

The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.

Under adverse environmental conditions bacterial cells survive by activating the “stringent response,” which is mediated by the accumulation of small signaling alarmone molecules (p)ppGpp. The synthesis of (p)ppGpp from ATP and GDP/GTP and their hydrolysis to GDP/GTP and pyrophosphate are catalyzed by enzymes of the RSH (RelA/SpoT Homologue) family. Studying the complex allosteric mechanism underlying the bifunctional activity of these enzymes and the maintenance of intracellular levels of (p)ppGpp is a topical issue in modern science. In this work we determine the conformational stability and binding efficiency of the bifunctional homologue of the RSH family Rel_Seq385 with nucleotides depending on their concentration and the ionic composition of the reaction mixture.

Bacterial translation initiation factor IF3 is composed of two distinct domains—a well-characterized C-terminal domain (IF3C), which enhances the speed and fidelity of translation initiation, and a less understood N-terminal domain (IF3N). In this study, we developed an aptamer (Apt ³⁴³ ) that targets IF3N with the goal of elucidating its contribution to translation initiation. Rapid kinetics assays revealed that Apt ³⁴³ reduces the rate of IF3 association with the 30S ribosomal subunit by 13-fold, while inducing a pronounced rearrangement of both IF3 domains on the 30S. These changes compromise IF2-, mRNA-, and fMet-tRNA ^fMet -dependent movements of IF3, delaying 30S initiation complex (30S IC) formation by up to two orders of magnitude. Cryo-EM analysis suggests that Apt ³⁴³ may sterically clash with fMet-tRNA ^fMet , thereby perturbing the canonical pathway by which the initiator tRNA is accommodated after IF2-dependent recruitment and prior to start codon decoding. However, once the 30S IC is formed, blocking IF3N does not prevent 50S subunit joining or 70S IC assembly. Collectively, these findings support a role for IF3N in enhancing an efficient path for fMet-tRNA ^fMet accommodation towards the 30S IC and promoting IF3C displacement to unlock 50S recruitment. Moreover, this aptamer-based strategy offers a valuable tool for dissecting domain-specific activities of multidomain factors within complex environments such as the initiating ribosome. GRAPHICAL

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDF E REGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDF G REGYS). It was also shown that the SGYDF E REGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction’s equilibrium dissociation constant is five orders of magnitude lower than for the SGYDF G REGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins was studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.