The structure and activities of the archaeal transcription termination factor Eta detail vulnerabilities of the transcription elongation complex.

Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.

Governance and the mangrove commons: Advancing the cross-scale, nested framework for the global conservation and wise use of mangroves.

Mangroves provide critical ecosystems services, contributing an estimated 42 billion US dollars to global fisheries, storing 25.5 million tons of carbon per year, and providing flood protection to over 15 million people annually. Yet, they are increasingly threatened by factors ranging from local resource exploitation to global climate change, with an estimated 35% of mangrove forests lost in the past two decades. These threats are difficult to manage due to the intrinsic characteristics of mangrove systems and their provisioning services, and their transboundary and pan-global nature. Due to their unique intertidal ecological niche, mangroves are often treated as a "common pool resource" within national legal frameworks, making them particularly susceptible to exploitation. Moreover, they form ecological connections through numerous biotic and abiotic processes that cross political boundaries. Because of these qualities a cross-scale nested framework of international, regional, and local coordination is necessary to successfully sustain mangrove ecosystems and their valuable services. Although coordination across the geopolitical spectrum is often cited as a need for effective management of common resources such as mangroves, there has been no formal analysis of mangrove multiscale governance. In this paper we address this gap by providing a comprehensive analysis of interactions between and within international, regional, and local mangrove management regimes and examine the challenges and opportunities such multiscale governance frameworks present. We highlight Costa Rica as a case study to demonstrate the universal relevance and potential of multi-scale governance and explore its downscale potential. Using Elinor Ostrom's principles for self-governance of the commons as our touchstone, we identify where improvements to the status quo could be implemented to increase its effectiveness of the current frameworks to meet the ongoing challenge of managing mangrove-derived resources and services in the face of a changing climate and human needs.

FttA is a CPSF73 homologue that terminates transcription in Archaea.

Regulated gene expression is largely achieved by controlling the activities of essential, multisubunit RNA polymerase transcription elongation complexes (TECs). The extreme stability required of TECs to processively transcribe large genomic regions necessitates robust mechanisms to terminate transcription. Efficient transcription termination is particularly critical for gene-dense bacterial and archaeal genomes1-3 in which continued transcription would necessarily transcribe immediately adjacent genes and result in conflicts between the transcription and replication apparatuses4-6; the coupling of transcription and translation7,8 would permit the loading of ribosomes onto aberrant transcripts. Only select sequences or transcription termination factors can disrupt the otherwise extremely stable TEC and we demonstrate that one of the last universally conserved archaeal proteins with unknown biological function is the Factor that terminates transcription in Archaea (FttA). FttA resolves the dichotomy of a prokaryotic gene structure (operons and polarity) and eukaryotic molecular homology (general transcription apparatus) that is observed in Archaea. This missing link between prokaryotic and eukaryotic transcription regulation provides the most parsimonious link to the evolution of the processing activities involved in RNA 3'-end formation in Eukarya.

Development of both type I-B and type II CRISPR/Cas genome editing systems in the cellulolytic bacterium Clostridium thermocellum.

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

TFS and Spt4/5 accelerate transcription through archaeal histone-based chromatin.

RNA polymerase must surmount translocation barriers for continued transcription. In Eukarya and most Archaea, DNA-bound histone proteins represent the most common and troublesome barrier to transcription elongation. Eukaryotes encode a plethora of chromatin-remodeling complexes, histone-modification enzymes and transcription elongation factors to aid transcription through nucleosomes, while archaea seemingly lack machinery to remodel/modify histone-based chromatin and thus must rely on elongation factors to accelerate transcription through chromatin-barriers. TFS (TFIIS in Eukarya) and the Spt4-Spt5 complex are universally encoded in archaeal genomes, and here we demonstrate that both elongation factors, via different mechanisms, can accelerate transcription through archaeal histone-based chromatin. Histone proteins in Thermococcus kodakarensis are sufficiently abundant to completely wrap all genomic DNA, resulting in a consistent protein barrier to transcription elongation. TFS-enhanced cleavage of RNAs in backtracked transcription complexes reactivates stalled RNAPs and dramatically accelerates transcription through histone-barriers, while Spt4-Spt5 changes to clamp-domain dynamics play a lesser-role in stabilizing transcription. Repeated attempts to delete TFS, Spt4 and Spt5 from the T. kodakarensis genome were not successful, and the essentiality of both conserved transcription elongation factors suggests that both conserved elongation factors play important roles in transcription regulation in vivo, including mechanisms to accelerate transcription through downstream protein barriers.

Factor-dependent archaeal transcription termination.

RNA polymerase activity is regulated by nascent RNA sequences, DNA template sequences, and conserved transcription factors. Transcription factors promoting initiation and elongation have been characterized in each domain, but transcription termination factors have been identified only in bacteria and eukarya. Here we describe euryarchaeal termination activity (Eta), the first archaeal termination factor capable of disrupting the transcription elongation complex (TEC), detail the rate of and requirements for Eta-mediated transcription termination, and describe a role for Eta in transcription termination in vivo. Eta-mediated transcription termination is energy-dependent, requires upstream DNA sequences, and disrupts TECs to release the nascent RNA to solution. Deletion of TK0566 (encoding Eta) is possible, but results in slow growth and renders cells sensitive to DNA damaging agents. Our results suggest that the mechanisms used by termination factors in archaea, eukarya, and bacteria to disrupt the TEC may be conserved, and that Eta stimulates release of stalled or arrested TECs.

Transcription Regulation in Archaea.

The known diversity of metabolic strategies and physiological adaptations of archaeal species to extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that gene expression patterns match the needs of the cell necessitate regulatory strategies that control the activities and output of the archaeal transcription apparatus. Archaea are reliant on a single RNA polymerase for all transcription, and many of the known regulatory mechanisms employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial species. Novel mechanisms of transcription regulation have become apparent by increasingly sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes recent progress in understanding archaeal transcription regulatory mechanisms and highlights insights gained from studies of the influence of archaeal chromatin on transcription.

Analyses of in vivo interactions between transcription factors and the archaeal RNA polymerase.

Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation.