RNase E and the High-Fidelity Orchestration of RNA Metabolism.

The bacterial endoribonuclease RNase E occupies a pivotal position in the control of gene expression, as its actions either commit transcripts to an irreversible fate of rapid destruction or unveil their hidden functions through specific processing. Moreover, the enzyme contributes to quality control of rRNAs. The activity of RNase E can be directed and modulated by signals provided through regulatory RNAs that guide the enzyme to specific transcripts that are to be silenced. Early in its evolutionary history, RNase E acquired a natively unfolded appendage that recruits accessory proteins and RNA. These accessory factors facilitate the activity of RNase E and include helicases that remodel RNA and RNA-protein complexes, and polynucleotide phosphorylase, a relative of the archaeal and eukaryotic exosomes. RNase E also associates with enzymes from central metabolism, such as enolase and aconitase. RNase E-based complexes are diverse in composition, but generally bear mechanistic parallels with eukaryotic machinery involved in RNA-induced gene regulation and transcript quality control. That these similar processes arose independently underscores the universality of RNA-based regulation in life. Here we provide a synopsis and perspective of the contributions made by RNase E to sustain robust gene regulation with speed and accuracy.Wellcome Trus

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support

Staphylococcus aureus sacculus mediates activities of M23 hydrolases.

International audiencePeptidoglycan, a gigadalton polymer, functions as the scaffold for bacterial cell walls and provides cell integrity. Peptidoglycan is remodelled by a large and diverse group of peptidoglycan hydrolases, which control bacterial cell growth and division. Over the years, many studies have focused on these enzymes, but knowledge on their action within peptidoglycan mesh from a molecular basis is scarce. Here, we provide structural insights into the interaction between short peptidoglycan fragments and the entire sacculus with two evolutionarily related peptidases of the M23 family, lysostaphin and LytM. Through nuclear magnetic resonance, mass spectrometry, information-driven modelling, site-directed mutagenesis and biochemical approaches, we propose a model in which peptidoglycan cross-linking affects the activity, selectivity and specificity of these two structurally related enzymes differently

Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy

International audienceThe rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of β-lactams by β-lactamases, (ii) the interaction of drugs belonging to the β-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm

Structural analysis of mtEXO mitochondrial RNA degradosome reveals tight coupling of nuclease and helicase components

The mitochondrial RNA degradosome (mtEXO) plays an essential role in the regulation of mitochondrial gene expression and is composed of the 3′-to-5′ exoribonuclease Dss1 and the helicase Suv3. Here the authors present the RNA bound mtEXO crystal structure and give insights into its mechanism

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

TET ten eleven translocation enzymes catalyze the oxidation of 5 methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix loop helix and basic leucine zipper domain transcription factor binding sites, with up to 250 fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation sensitive immediate early transcription factors and octamer binding transcription factor 4 OCT4 , respectively, illuminating TET function in transcriptional responses and pluripotency suppor