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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 harbor 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 transposasons. 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 HTH 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.

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 MJ1647 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 like M. jannaschii A3, 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 compacts 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. Our results reveal MJ1647 as the first histone with DNA bridging properties, which has important implications for genome structure and gene expression in archaea.

The mining of genomes from non-cultivated microorganisms using metagenomics is a powerful tool to discover novel proteins and other valuable biomolecules. However, function-based metagenome searches are often limited by the time-consuming expression of the active proteins in various heterologous host systems. We here report the initial characterization of novel single-subunit bacteriophage RNA polymerase, RNAP_E, identified from a metagenome data set obtained from an elephant dung microbiome. RNAP_E and its promoter sequence are distantly related to T7 RNA polymerase. Using RNAP_E and a translation-competent Escherichia coli extract, we have developed an efficient medium throughput pipeline and protocol allowing the expression of metagenome-derived genes and the production of proteins in cell-free system is sufficient for the initial testing of the predicted activities. Here, we have successfully identified and verified 12 enzymes acting on bis(2-hydroxyethyl) terephthalate (BHET) in a completely clone-free approach and proposed an in vitro high-throughput metagenomic screening method with RNAP_E.

Recent developments in electron-transparent materials have paved the way for liquid-phase electron microscopy (LPEM) leading to an unprecedented understanding of the structure and dynamics of specimens in their liquid environment. Image reconstruction in liquid-state poses several challenges, and most importantly, it undermines the single-particle analysis assumption that the three-dimensional objects captured on the image sensor are identical. We propose the combination of all-atom simulations with LPEM to complement structural studies with dynamic investigations. In this work, we exploited LPEM to image the dynamics of particles undergoing Brownian motion, using their natural rotation to access the particle structural landscape for reconstructing its architecture in 3D using tomographic techniques. We have selected two test cases for our approach according to prior data accessibility and physiological environment factors: apoferritin and archaeal RNA polymerase. We show that the adopted approach allows to achieve sub-nanometer spatial resolutions of protein structures, either imaging proteins one by one and assessing different conformational states or combining several proteins into one statistical conformational ensemble.