DNA-bridging by an archaeal histone variant via a unique tetramerisation interface

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into 'hypernucleosome' particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea

Function of the trigger loop in distinct steps of the transcription cycle

RNA polymerases (RNAPs) carry out transcription in all living organisms. Whereas only one RNAP is present in bacteria and archaea, eukaryotes possess three to five specialized nuclear RNA polymerases (RNAP I, II, III, IV and V). To date, various RNAP structures have elucidated functional mechanisms of transcription, but biochemical analysis on eukaryotic RNAP II mutants are generally restricted to viable yeast strains. The active site of the RNAP catalyzes RNA chain growth by phosphodiester bond formation. The active site of all RNAPs contains two Mg2+ ions and an evolutionarily conserved mobile element, the trigger loop (TL) that functions in the elongation phase of transcription. Employing the reconstituted archaeal in vitro transcription system of Pyrococcus furiosus, we analyzed deletion mutants of the trigger loop and introduced alanine substitutions at key residues A'' Q80, A'' L83 and A'' H87. Furthermore, four supplementary mutants were analyzed in order to clarify their participation in NTP/2'dNTP discrimination. The work of this thesis reveals that the archaeal TL is absolutely essential for transcription initiation, specially for capturing the incoming NTPs before a stable DNA–RNA hybrid is present in the active centre. Moreover, catalysis in initially transcribing complexes, active site closure and synthesis stimulation by the TL were crucial, as A'' H87 and the TLtip region were essential for initiation. In vitro elongation assays also provided insights into TL function during the discrimination of correct NTPs from wrong dNTPs. Although A'' L83 and A'' H87 contribute to the recognition of the correct NTP and to the catalysis, A'' Q80 contributes to the recognition of the 2′OH-group of the NTP, indicating the critical role of the interaction between the TL and the NTP for the nucleotide incorporation fidelity. Transcription fidelity also relies on proofreading, a post-incorporation mechanism that involves cleavage of a dinucleotide from the RNA 3′-end containing the misincorporated nucleotide. The TL is not required for the intrinsic cleavage activity of the enzyme but influences the translocation by being part of the Brownian ratchet that underlies translocation. The data shown here also suggests the critical role of the residue A'' L83 in backtracked complex stability. Proofreading is stimulated by extrinsic RNA cleavage factors such as TFS, and we found that the TL is also dispensable for TFS-stimulated cleavage, consistent with structures illustrating that the TL is in the locked conformation in the presence of the eukaryotic TFS counterpart, TFIIS. Finally, our results revealed a function of the TL in transcription termination. We show that the sensitivity of the archaeal RNAP to poly-T sequences is increased on TL truncation, showing that the TL prevents aberrant termination at non-terminator sites, to ensure processive RNA synthesis

The RNA polymerase trigger loop functions in all three phases of the transcription cycle

The trigger loop (TL) forms a conserved element in the RNA polymerase active centre that functions in the elongation phase of transcription. Here, we show that the TL also functions in transcription initiation and termination. Using recombinant variants of RNA polymerase from Pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the TL is essential for initial RNA synthesis until a complete DNA–RNA hybrid is formed. The archaeal TL is further important for transcription fidelity during nucleotide incorporation, but not for RNA cleavage during proofreading. A conserved glutamine residue in the TL binds the 2’-OH group of the nucleoside triphosphate (NTP) to discriminate NTPs from dNTPs. The TL also prevents aberrant transcription termination at non-terminator sites

Cbp1 and Cren7 form chromatin-like structures that ensure efficient transcription of long CRISPR arrays

Abstract CRISPR arrays form the physical memory of CRISPR adaptive immune systems by incorporating foreign DNA as spacers that are often AT-rich and derived from viruses. As promoter elements such as the TATA-box are AT-rich, CRISPR arrays are prone to harbouring cryptic promoters. Sulfolobales harbour extremely long CRISPR arrays spanning several kilobases, a feature that is accompanied by the CRISPR-specific transcription factor Cbp1. Aberrant Cbp1 expression modulates CRISPR array transcription, but the molecular mechanisms underlying this regulation are unknown. Here, we characterise the genome-wide Cbp1 binding at nucleotide resolution and characterise the binding motifs on distinct CRISPR arrays, as well as on unexpected non-canonical binding sites associated with transposons. Cbp1 recruits Cren7 forming together ‘chimeric’ chromatin-like structures at CRISPR arrays. We dissect Cbp1 function in vitro and in vivo and show that the third helix-turn-helix domain is responsible for Cren7 recruitment, and that Cbp1-Cren7 chromatinization plays a dual role in the transcription of CRISPR arrays. It suppresses spurious transcription from cryptic promoters within CRISPR arrays but enhances CRISPR RNA transcription directed from their cognate promoters in their leader region. Our results show that Cbp1-Cren7 chromatinization drives the productive expression of long CRISPR arrays

Structural basis of RNA polymerase inhibition by viral and host factors

International audienceRNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition

A performance evaluation and inter-laboratory comparison of community face coverings media in the context of covid-19 pandemic

International audienceDuring the recent pandemic of SARS-CoV-2, and as a reaction to the worldwide shortage of surgical masks, several countries have introduced new types of masks named “community face covering” (CoFC). To ensure the quality of such devices and their relevance to slow down the virus spreading, a quick reaction of the certification organisms was necessary to fix the minimal acceptable performances requirements. Moreover, many laboratories involved in the aerosol research field have been asked to perform tests in a quick time according to (CEN, 2020) proposed by the European committee for standardization. This specification imposes a minimal air permeability of 96 L.m-2.s-1 and a minimal filtration efficiency of 70% for 3 µm diameter particles. In the present article, an intercomparison of efficiency and permeability measured by 3 testing laboratories has been performed. Results are in good agreement considering the heterogeneity of the considered material samples (within 27 % in terms of filtration efficiency and less than 20 % in terms of permeability). On this basis, an analysis of 233 materials made of woven, non-woven and mixed fibrous material has been done in terms of filtration efficiency and air permeability. For some of them, measurements have been performed for 0.2 µm, 1 µm and 3 µm particle diameters. As expected, no deterministic correlation could be determinated to link these efficiencies to the permeability of the considered samples; however, a trend could be identified. The same exercise has been conducted to link the filtration efficiency measured at 3 µm to the filtration for lower diameters. Finally, a discussion on the kind of material that is the most relevant to manufacture “community face covering” (CoFC) supported by spectral filtration efficiency (from 0.02 µm to 3 µm) is proposed

An Extended Network of Genomic Maintenance in the Archaeon Pyrococcus abyssi Highlights Unexpected Associations between Eucaryotic Homologs

In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes

DNA-bridging by an archaeal histone variant via a unique tetramerisation interface

Abstract In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into ‘hypernucleosome’ particles of varying sizes with each dimer wrapping 30 bp of DNA. These are composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension enabling homotetramerisation. The 1.9 Å X-ray structure of a dimeric MJ1647 species, structural modelling of the tetramer, and site-directed mutagenesis reveal that the C-terminal tetramerization module consists of two alpha helices in a handshake arrangement. Unlike canonical histones, MJ1647 tetramers can bridge two DNA molecules in vitro. Using single-molecule tethered particle motion and DNA binding assays, we show that MJ1647 tetramers bind ~60 bp DNA and compact DNA in a highly cooperative manner. We furthermore show that MJ1647 effectively competes with the transcription machinery to block access to the promoter in vitro. To the best of our knowledge, MJ1647 is the first histone shown to have DNA bridging properties, which has important implications for genome structure and gene expression in archaea