Biology subjects

Milon, P.

Publications and source records attributed to Milon, P..

3 recordsLinked to original sources

bioRxiv2019

The complete, functional and dynamic cycle of the bacterial Initiation Factor 3

Initiation factor 3 (IF3) is an essential protein that enhances the fidelity and speed of bacterial initiation of mRNA translation. The dynamic interplay between the two independent IF3 domains, their alternative binding sites, and the mechanism that ensures translation initiation fidelity remains elusive. Here, we show that the functional positioning of IF3 domains occurs at velocities ranging over two orders of magnitude, driven by each 30S initiation ligand. IF1 and IF2 rapidly promote the accommodation of IF3 on the 30S platform with the C-terminal domain moving towards the P site. Reversion of this movement is triggered by decoding the mRNA start codon and rate limits translation initiation. Binding of the tRNA results in the concomitant accommodation of the N-terminal domain of IF3, largely dependent on the mRNA and initiator tRNA. 70S initiation complex formation promotes the closing and dissociation of IF3, recycling the factor for a new round of translation initiation. Altogether our results unveil the kinetic spectrum of IF3 conformations and highlight fundamental movements of the factor that ensure accurate translation initiation.

biophysics↗

bioRxiv2019

How the initiating ribosome copes with (p)ppGpp to translate mRNAs

During host colonization, bacteria use the alarmone (p)ppGpp to reshape its proteome by acting pleiotropically on RNA and protein synthesis. Here, we elucidate how the translation Initiation Factor 2 (IF2) senses the cellular ppGpp to GTP ratio and regulates the progression towards protein synthesis. Our results show that the affinity of GTP and the inhibitory concentration of ppGpp for 30S-bound IF2 vary depending on the programmed mRNA. Highly translated mRNAs enhanced GTP affinity for 30S complexes, resulting in fast transitions to elongation of protein synthesis. Less demanded mRNAs allowed ppGpp to compete with GTP for IF2, stalling 30S complexes until exchange of the mRNA enhances the affinity for GTP. 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 its proteome as a function of the nutritional shift happening during infection.

biochemistry↗

bioRxiv2019

DNA aptamers for the recognition of HMGB1 from Plasmodium falciparum

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable and steadily present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in less than 30 seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a potential biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.

bioengineering↗