aCPSF1 cooperates with terminator U-tract to dictate archaeal transcription termination efficacy

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea; however, the working mechanism remains elusive. This work, through analyzing transcript-3′end-sequencing data of Methanococcus maripaludis, found genome-wide positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficacies (TTEs). In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding affinity, and in vivo assays demonstrated the two elements are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract cooperatively determine high TTEs. The N-terminal KH domains equip aCPSF1 with specific-binding capacity to terminator U-tract and the aCPSF1-terminator U-tract cooperation; while the nuclease activity of aCPSF1 was also required for TTEs. aCPSF1 also guarantees the terminations of transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract cooperation in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in archaea, which may be widely employed by archaeal phyla; using one trans-action factor to recognize U-rich terminator signal and cleave transcript 3′-end, the archaeal aCPSF1-dependent transcription termination may represent a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

Taxon-Specific Shifts in Bacterial and Archaeal Transcription of Dissolved Organic Matter Cycling Genes in a Stratified Fjord

It is generally recognized that stratification in the ocean strongly influences both the community composition and the distribution of ecological functions of microbial communities, which in turn are expected to shape the biogeochemical cycling of essential elements over depth. Here, we used metatranscriptomics analysis to infer molecular detail on the distribution of gene systems central to the utilization of organic matter in a stratified marine system.

Single-Molecule/Cell Analyses Reveal Principles of Genome-Folding Mechanisms in the Three Domains of Life

Comparative structural/molecular biology by single-molecule analyses combined with single-cell dissection, mass spectroscopy, and biochemical reconstitution have been powerful tools for elucidating the mechanisms underlying genome DNA folding. All genomes in the three domains of life undergo stepwise folding from DNA to 30–40 nm fibers. Major protein players are histone (Eukarya and Archaea), Alba (Archaea), and HU (Bacteria) for fundamental structural units of the genome. In Euryarchaeota, a major archaeal phylum, either histone or HTa (the bacterial HU homolog) were found to wrap DNA. This finding divides archaea into two groups: those that use DNA-wrapping as the fundamental step in genome folding and those that do not. Archaeal transcription factor-like protein TrmBL2 has been suggested to be involved in genome folding and repression of horizontally acquired genes, similar to bacterial H-NS protein. Evolutionarily divergent SMC proteins contribute to the establishment of higher-order structures. Recent results are presented, including the use of Hi-C technology to reveal that archaeal SMC proteins are involved in higher-order genome folding, and the use of single-molecule tracking to reveal the detailed functions of bacterial and eukaryotic SMC proteins. Here, we highlight the similarities and differences in the DNA-folding mechanisms in the three domains of life.

The Archaeal Transcription Termination Factor aCPSF1 is a Robust Phylogenetic Marker for Archaeal Taxonomy

Archaea represent a unique type of prokaryote, which inhabit in various environments including extreme environments, and so define the boundary of biosphere, and play pivotal ecological roles, particularly in extreme environments. Since their discovery over 40 years ago, environmental archaea have been widely investigated using the 16S rRNA sequence comparison, and the recently developed phylogenomic approach because the majority of archaea are recalcitrant to laboratory cultivation.

Taxon-specific shifts in bacterial and archaeal transcription of dissolved organic matter cycling genes in a stratified fjord

A considerable fraction of organic matter derived from photosynthesis in the euphotic zone settles into the ocean's interior, and under way is degraded by diverse microbial consortia that utilize a suite of extracellular enzymes and membrane transporters. Still, the molecular details that regulate carbon cycling across depths remain little explored. As stratification in fjords has made them attractive models to explore patterns in biological oceanography, we here analyzed bacterial and archaeal transcription in samples from five depth layers in the Gullmar Fjord, Sweden. Transcriptional variation over depth correlated with gradients in chlorophyll a and nutrient concentrations. Differences in transcription between sampling dates (summer and early autumn), were strongly correlated with ammonium concentrations, which potentially was linked with a stronger influence of (micro-)zooplankton grazing in summer. Transcriptional investment in carbohydrate-active enzymes (CAZymes) decreased with depth and shifted toward peptidases, partly a result of elevated CAZyme transcription by Flavobacteriales, Cellvibrionales and Synechococcales at 2-25 m and a dominance of peptidase transcription by Alteromonadales and Rhodobacterales from 50 m and down. In particular, CAZymes for chitin, laminarin, and glycogen were important. High levels of transcription of ammonium transporters by Thaumarchaeota at depth (up to 18% of total transcription), along with the genes for ammonia oxidation and CO2-fixation, indicated that chemolithoautotrophy contributed to the carbon flux in the fjord. The taxon-specific expression of functional genes for processing of the marine DOM pool and nutrients across depths emphasizes the importance of different microbial foraging mechanisms across spatiotemporal scales for shaping biogeochemical cycles.

aCPSF1 in synergy with terminator U-tract dictates archaeal transcription termination efficiency via the KH domains recognizing U-tract

Recently, aCPSF1 was reported to function as the long-sought global transcription termination factor of archaea, while the working mechanism remains elusive. This work, through analyzing transcript-3′end-sequencing data of Methanococcus maripaludis, found positive correlations of both the terminator uridine(U)-tract and aCPSF1 with hierarchical transcription termination efficiencies (TTEs) at the genome-wide level. In vitro assays determined that aCPSF1 specifically binds to the terminator U-tract with U-tract number-related binding abilities, and in vivo assays demonstrated the two are indispensable in dictating high TTEs, revealing that aCPSF1 and the terminator U-tract in synergy determine high TTEs. The N-terminal KH domains equip aCPSF1 of specific binding to terminator U-tract and the in vivo aCPSF1-terminator U-tract synergism; aCPSF1's nuclease activity was also required for TTEs. aCPSF1 also functioned as back-up termination for transcripts with weak intrinsic terminator signals. aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota exhibited similar U-tract synergy in dictating TTEs. Therefore, aCPSF1 and the intrinsic U-rich terminator could work in a noteworthy two-in-one termination mode in Archaea, which could be widely employed by archaeal phyla; using one factor recognizing U-rich terminator signal and cleaving transcript 3′-end, the archaeal aCPSF1-dependent transcription termination could display a simplified archetypal mode of the eukaryotic RNA polymerase II termination machinery.

The conserved ribonuclease aCPSF1 triggers genome-wide transcription termination of Archaea via a 3′-end cleavage mode

Transcription termination defines accurate transcript 3′-ends and ensures programmed transcriptomes, making it critical to life. However, transcription termination mechanisms remain largely unknown in Archaea. Here, we reported the physiological significance of the newly identified general transcription termination factor of Archaea, the ribonuclease aCPSF1, and elucidated its 3′-end cleavage triggered termination mechanism. The depletion of Mmp-aCPSF1 in Methanococcus maripaludis caused a genome-wide transcription termination defect and disordered transcriptome. Transcript-3′end-sequencing revealed that transcriptions primarily terminate downstream of a uridine-rich motif where Mmp-aCPSF1 performed an endoribonucleolytic cleavage, and the endoribonuclease activity was determined to be essential to the in vivo transcription termination. Co-immunoprecipitation and chromatin-immunoprecipitation detected interactions of Mmp-aCPSF1 with RNA polymerase and chromosome. Phylogenetic analysis revealed that the aCPSF1 orthologs are ubiquitously distributed among the archaeal phyla, and two aCPSF1 orthologs from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to terminate transcription of M. maripaludis. Therefore, the aCPSF1 dependent termination mechanism could be widely employed in Archaea, including Lokiarchaeota belonging to Asgard Archaea, the postulated archaeal ancestor of Eukaryotes. Strikingly, aCPSF1-dependent archaeal transcription termination reported here exposes a similar 3′-cleavage mode as the eukaryotic RNA polymerase II termination, thus would shed lights on understanding the evolutionary linking between archaeal and eukaryotic termination machineries.

aCPSF1 controlled archaeal transcription termination: a prototypical eukaryotic model

Transcription termination defines RNA 3′-ends and guarantees programmed transcriptomes, thus is an essential biological process for life. However, transcription termination mechanisms remain almost unknown in Archaea. Here reported the first general transcription termination factor of Archaea, the conserved ribonuclease aCPSF1, and elucidated its 3′-end cleavage dependent termination mechanism. Depletion of Mmp-aCPSF1 in a methanoarchaeon Methanococcus maripaludis caused a genome-wide transcription termination defect and overall transcriptome chaos, and cold-sensitive growth. Transcript-3′end-sequencing (Term-seq) revealed transcriptions mostly terminated downstream of a uridine-rich terminator motif, where Mmp-aCPSF1 performed cleavage. The endoribonuclease activity was determined essential to terminate transcription in vivo as well. Through super-resolution photoactivated localization microscopy imaging, co-immunoprecipitation, and chromatin immunoprecipitation, we demonstrated that Mmp-aCPSF1 localizes within nucleoid and associates with RNAP and chromosomes. aCPSF1 appears to co-evolve with archaeal RNAPs, and two distant orthologs each from Lokiarchaeota and Thaumarchaeota could replace Mmp-aCPSF1 to termination transcription. Thus, aCPSF1 dependent termination mechanism could be universally employed in Archaea, including Lokiarchaeota, one supposed archaeal ancestor of Eukaryotes. Therefore, the reported aCPSF1 cleavage-dependent termination mode not only hints an archetype of Eukaryotic 3′-end processing/cleavage triggered RNAP II termination, but also would shed lights on understanding the complex eukaryotic termination based on the simplified archaeal model.