Biology subjects

Ennifar, E.

Publications and source records attributed to Ennifar, E..

3 recordsLinked to original sources

bioRxiv2021

The RNA chaperone protein CspA stimulates translation during cold acclimation by promoting the progression of the ribosomes

CspA is an RNA binding protein expressed during cold-shock in Escherichia coli, capable of stimulating translation of several mRNAs - including its own - at low temperature. We used reconstituted translation systems to monitor the effects of CspA on the different steps of the translation process and probing experiments to analyze the interactions with its target mRNAs. We specifically focused on cspA mRNA which adopts a cold-induced secondary structure at temperatures below 20{degrees}C and a more closed conformation at 37{degrees}C. We show that at low temperature CspA specifically promotes the translation of the mRNA folded in the conformation less accessible to the ribosome (37{degrees}C form). CspA interacts with its mRNA without inducing large structural rearrangement, does not bind the ribosomal subunits and is not able to stimulate the formation of the translation initiation complexes. On the other hand, CspA promotes the progression of the ribosomes during translation of its mRNA at low temperature and this stimulation is mRNA structure-dependent. A similar structure-dependent mechanism may be responsible for the CspA- dependent translation stimulation observed with other probed mRNAs, for which the transition to the elongation phase is progressively facilitated during cold acclimation with the accumulation of CspA.

molecular biology↗

bioRxiv2020

Safe and easy evaluation of tmRNA-SmpB-mediated trans-translation in ESKAPE pathogenic bacteria

Bacteria cope with ribosome stalling thanks to trans-translation, a major quality control system of protein synthesis that is mediated by tmRNA, an hybrid RNA with properties of both a tRNA and an mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, none of these permits the safe and easy evaluation of trans-translation in pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens.

biochemistry↗

bioRxiv2020

Deflating the RNA Mg2+ bubble. Stereochemistry to the rescue!

Proper evaluation of the ionic structure of biomolecular systems remains challenging in X-ray and cryo-EM techniques but is essential for advancing our understanding of complex structure/activity/solvent relationships. However, numerous studies overestimate the number of Mg2+ in the deposited structures and underrate the importance of stereochemical rules to correctly assign these ions. Herein, we re-evaluate the PDBid 6QNR and 6SJ6 models of the ribosome ionic structure and establish that stereochemical principles should always be considered when evaluating ion binding features, even when K+ anomalous signals are available as it is the case for 6QNR. Assignment errors can result in misleading conceptions of the solvent structure of ribosomes and other RNA systems and should therefore be avoided. Our analysis resulted in a significant decrease of bound Mg2+ ions in the 6QNR structure, suggesting that K+ and not Mg2+ is the prevalent ion in the ribosome 1st solvation shell. We stress that the use of proper stereochemical guidelines is critical for deflating the current Mg2+ bubble witnessed in many ribosome and other RNA structures. Herewith, we would like to draw the attention of the researchers interested in the ionic structure of biomolecular systems on the importance and complementarity of stereochemistry and other ion identification techniques such as those pertaining to the detection of anomalous signals of transition metals and K+. We also stress that for the identification of lighter ions such as Mg2+, Na+, ..., stereochemistry coupled with high resolution structures remain the best currently available option.

biochemistry↗