RESUMEN
RNAs are often modified to invoke new activities. While many modifications are limited in frequency, restricted to non-coding RNAs, or present only in select organisms, 5-methylcytidine (m5C) is abundant across diverse RNAs and fitness-relevant across Domains of life, but the synthesis and impacts of m5C have yet to be fully investigated. Here, we map m5C in the model hyperthermophile, Thermococcus kodakarensis. We demonstrate that m5C is ~25x more abundant in T. kodakarensis than human cells, and the m5C epitranscriptome includes ~10% of unique transcripts. T. kodakarensis rRNAs harbor tenfold more m5C compared to Eukarya or Bacteria. We identify at least five RNA m5C methyltransferases (R5CMTs), and strains deleted for individual R5CMTs lack site-specific m5C modifications that limit hyperthermophilic growth. We show that m5C is likely generated through partial redundancy in target sites among R5CMTs. The complexity of the m5C epitranscriptome in T. kodakarensis argues that m5C supports life in the extremes.
Asunto(s)
Citidina , Metiltransferasas , Thermococcus , Transcriptoma , Thermococcus/genética , Thermococcus/metabolismo , Thermococcus/enzimología , Metiltransferasas/metabolismo , Metiltransferasas/genética , Citidina/metabolismo , Citidina/análogos & derivados , Citidina/genética , Humanos , ARN de Archaea/genética , ARN de Archaea/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Arqueales/genética , ARN Ribosómico/metabolismo , ARN Ribosómico/genéticaRESUMEN
Many archaea encode and express histone proteins to compact their genomes. Archaeal and eukaryotic histones share a near-identical fold that permits DNA wrapping through select histone-DNA contacts to generate chromatin-structures that must be traversed by RNA polymerase (RNAP) to generate transcripts. As archaeal histones can spontaneously assemble with a single histone isoform, single-histone chromatin variants provide an idealized platform to detail the impacts of distinct histone-DNA contacts on transcription efficiencies and to detail the role of the conserved cleavage stimulatory factor, Transcription Factor S (TFS), in assisting RNAP through chromatin landscapes. We demonstrate that substitution of histone residues that modify histone-DNA contacts or the three-dimensional chromatin structure result in radically altered transcription elongation rates and pausing patterns. Chromatin-barriers slow and pause RNAP, providing regulatory potential. The modest impacts of TFS on elongation rates through chromatin landscapes is correlated with TFS-dispensability from the archaeon Thermococcus kodakarensis. Our results detail the importance of distinct chromatin structures for archaeal gene expression and provide a unique perspective on the evolution of, and regulatory strategies imposed by, eukaryotic chromatin.
Asunto(s)
Histonas , Thermococcus , Histonas/metabolismo , ADN de Archaea/genética , Cromatina/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Thermococcus/genética , Thermococcus/metabolismoRESUMEN
The sole unifying feature of the incredibly diverse Archaea is their isoprenoid-based ether-linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane-spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth in T. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra-optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid-based lipid membranes for archaeal survival at high temperatures.
Asunto(s)
Lípidos de la Membrana , Thermococcus , Lípidos de la Membrana/metabolismo , Thermococcus/metabolismo , Thermococcus/genética , Éteres de Glicerilo/metabolismo , Proteínas Arqueales/metabolismo , Archaea/metabolismo , Lípidos/químicaRESUMEN
Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , Thermococcus , Desoxirribonucleasa IV (Fago T4-Inducido) , Daño del ADN , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Thermococcus/enzimología , Thermococcus/genéticaRESUMEN
The ribonuclease FttA mediates factor-dependent transcription termination in archaea 1-3 . Here, we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'â3' exonucleolytic cleavage of RNA by FttA and concomitant 5'â3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, reveal functional homology between archaeal and eukaryotic factor-dependent termination, and reveal fundamental mechanistic similarities in factor-dependent termination in the three domains of life: bacterial, archaeal, and eukaryotic. One sentence summary: Cryo-EM reveals the structure of the archaeal FttA pre-termination complex.
RESUMEN
CRISPR-Cas systems provide heritable immunity against viruses and other mobile genetic elements by incorporating fragments of invader DNA into the host CRISPR array as spacers. Integration of new spacers is localized to the 5' end of the array, and in certain Gram-negative Bacteria this polarized localization is accomplished by the integration host factor. For most other Bacteria and Archaea, the mechanism for 5' end localization is unknown. Here we show that archaeal histones play a key role in directing integration of CRISPR spacers. In Pyrococcus furiosus, deletion of either histone A or B impairs integration. In vitro, purified histones are sufficient to direct integration to the 5' end of the CRISPR array. Archaeal histone tetramers and bacterial integration host factor induce similar U-turn bends in bound DNA. These findings indicate a co-evolution of CRISPR arrays with chromosomal DNA binding proteins and a widespread role for binding and bending of DNA to facilitate accurate spacer integration.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Histonas , Histonas/genética , Archaea/genética , Factores de Integración del Huésped , ADN , BacteriasRESUMEN
Genetic manipulation is an essential tool to investigate complex microbiological phenomena. In this chapter we describe the techniques required to transform the model hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis. T. kodakarensis can support two modes of genetic manipulation, dependent either on homologous recombination into the genome or through retention of autonomously replicating plasmids. The robust genetic system developed in T. kodakarensis offers a variety of selectable and counterselectable markers for complex, accurate and iterative genetic manipulations offering greater flexibility to probe gene function in vivo.
Asunto(s)
Thermococcus , Anaerobiosis , Plásmidos/genética , Thermococcus/genéticaRESUMEN
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.
Asunto(s)
Proteínas Arqueales , ADN Helicasas , Thermococcus , Factores de Transcripción , Terminación de la Transcripción Genética , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cristalografía , ADN Helicasas/química , ADN Helicasas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Dominios Proteicos , Thermococcus/enzimología , Thermococcus/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Hyperthermophiles, typically defined as organisms with growth optima ≥80°C, are dominated by the Archaea. Proteins that support life at the extremes of temperatures often retain substantial biotechnological and commercial value, but the recombinant expression of individual hyperthermophilic proteins is commonly complicated in non-native mesophilic hosts due to differences in codon bias, intracellular solutes and the requirement for accessory factors that aid in folding or deposition of metal centers within archaeal proteins. The development of versatile protein expression and facilitated protein purification systems in the model, genetically tractable, hyperthermophilic marine archaeon Thermococcus kodakarensis provides an attractive platform for protein expression within the hyperthermophiles. The assortment of T. kodakarensis genetic backgrounds and compatible selection markers allow iterative genetic manipulations that facilitate protein overexpression and expedite protein purifications. Expression vectors that stably replicate both in T. kodakarensis and Escherichia coli have been validated and permit high-level ectopic gene expression from a variety of controlled and constitutive promoters. Biologically relevant protein associations can be maintained during protein purifications to identify native protein partnerships and define protein interaction networks. T. kodakarensis thus provides a versatile platform for the expression and purification of thermostable proteins.
Asunto(s)
Proteínas Arqueales , Thermococcus , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas Genéticas , Temperatura , Thermococcus/genéticaRESUMEN
Thermococcus kodakarensis (T. kodakarensis), a hyperthermophilic, genetically accessible model archaeon, encodes two putative restriction modification (R-M) defense systems, TkoI and TkoII. TkoI is encoded by TK1460 while TkoII is encoded by TK1158. Bioinformative analysis suggests both R-M enzymes are large, fused methyltransferase (MTase)-endonuclease polypeptides that contain both restriction endonuclease (REase) activity to degrade foreign invading DNA and MTase activity to methylate host genomic DNA at specific recognition sites. In this work, we demonsrate T. kodakarensis strains deleted for either or both R-M enzymes grow more slowly but display significantly increased competency compared to strains with intact R-M systems, suggesting that both TkoI and TkoII assist in maintenance of genomic integrity in vivo and likely protect against viral- or plasmid-based DNA transfers. Pacific Biosciences single molecule real-time (SMRT) sequencing of T. kodakarensis strains containing both, one or neither R-M systems permitted assignment of the recognition sites for TkoI and TkoII and demonstrated that both R-M enzymes are TypeIIL; TkoI and TkoII methylate the N6 position of adenine on one strand of the recognition sequences GTGAAG and TTCAAG, respectively. Further in vitro biochemical characterization of the REase activities reveal TkoI and TkoII cleave the DNA backbone GTGAAG(N)20/(N)18 and TTCAAG(N)10/(N)8, respectively, away from the recognition sequences, while in vitro characterization of the MTase activities reveal transfer of tritiated S-adenosyl methionine by TkoI and TkoII to their respective recognition sites. Together these results demonstrate TkoI and TkoII restriction systems are important for protecting T. kodakarensis genome integrity from invading foreign DNA.
RESUMEN
Histone proteins compact and organize DNA resulting in a dynamic chromatin architecture impacting DNA accessibility and ultimately gene expression. Eukaryotic chromatin landscapes are structured through histone protein variants, epigenetic marks, the activities of chromatin-remodeling complexes, and post-translational modification of histone proteins. In most Archaea, histone-based chromatin structure is dominated by the helical polymerization of histone proteins wrapping DNA into a repetitive and closely gyred configuration. The formation of the archaeal-histone chromatin-superhelix is a regulatory force of adaptive gene expression and is likely critical for regulation of gene expression in all histone-encoding Archaea. Single amino acid substitutions in archaeal histones that block formation of tightly packed chromatin structures have profound effects on cellular fitness, but the underlying gene expression changes resultant from an altered chromatin landscape have not been resolved. Using the model organism Thermococcus kodakarensis, we genetically alter the chromatin landscape and quantify the resultant changes in gene expression, including unanticipated and significant impacts on provirus transcription. Global transcriptome changes resultant from varying chromatin landscapes reveal the regulatory importance of higher-order histone-based chromatin architectures in regulating archaeal gene expression.
RESUMEN
Archaea often thrive in environmental extremes, enduring levels of heat, pressure, salinity, pH, and radiation that prove intolerable to most life. Many environmental extremes raise the propensity for DNA damaging events and thus, impact DNA stability, placing greater reliance on molecular mechanisms that recognize DNA damage and initiate accurate repair. Archaea can presumably prosper in harsh and DNA-damaging environments in part due to robust DNA repair pathways but surprisingly, no DNA repair pathways unique to Archaea have been described. Here, we review the most recent advances in our understanding of archaeal DNA repair. We summarize DNA damage types and their consequences, their recognition by host enzymes, and how the collective activities of many DNA repair pathways maintain archaeal genomic integrity.
Asunto(s)
Archaea/genética , ADN de Archaea/genética , Daño del ADN , Reparación del ADNRESUMEN
Increasingly sophisticated biochemical and genetic techniques are unraveling the regulatory factors and mechanisms that control gene expression in the Archaea. While some similarities in regulatory strategies are universal, archaeal-specific regulatory strategies are emerging to complement a complex patchwork of shared archaeal-bacterial and archaeal-eukaryotic regulatory mechanisms employed in the archaeal domain. The prokaryotic archaea encode core transcription components with homology to the eukaryotic transcription apparatus and also share a simplified eukaryotic-like initiation mechanism, but also deploy tactics common to bacterial systems to regulate promoter usage and influence elongation-termination decisions. We review the recently established complete archaeal transcription cycle, highlight recent findings of the archaeal transcription community and detail the expanding post-initiation regulation imposed on archaeal transcription.
Asunto(s)
Archaea/genética , Transcripción Genética/genéticaRESUMEN
N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.
Asunto(s)
Acetilación , Citidina/análogos & derivados , Células Eucariotas/metabolismo , Evolución Molecular , ARN/química , ARN/metabolismo , Archaea/química , Archaea/citología , Archaea/genética , Archaea/crecimiento & desarrollo , Secuencia Conservada , Microscopía por Crioelectrón , Citidina/metabolismo , Células Eucariotas/citología , Células HeLa , Humanos , Modelos Moleculares , Acetiltransferasas N-Terminal/metabolismo , ARN de Archaea/química , ARN de Archaea/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Ribosomas/ultraestructura , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , TemperaturaRESUMEN
Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.
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Guanosina/análogos & derivados , Methanosarcina/metabolismo , ARN de Archaea/genética , ARN de Transferencia/genética , Thermococcus/metabolismo , Guanosina/metabolismo , Methanosarcina/química , Methanosarcina/genética , Estabilidad del ARN , ARN de Archaea/química , ARN de Archaea/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Thermococcus/química , Thermococcus/genéticaRESUMEN
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.
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Proteínas Arqueales/química , Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Genoma Arqueal , Thermococcus/genética , Factores de Transcripción/química , Terminación de la Transcripción Genética , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Bacterias/genética , Bacterias/metabolismo , Evolución Biológica , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Humanos , Modelos Moleculares , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología Estructural de Proteína , Thermococcus/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Iniciación de la Transcripción GenéticaRESUMEN
Reactive oxygen species drive the oxidation of guanine to 8-oxoguanine (8oxoG), which threatens genome integrity. The repair of 8oxoG is carried out by base excision repair enzymes in Bacteria and Eukarya, however, little is known about archaeal 8oxoG repair. This study identifies a member of the Ogg-subfamily archaeal GO glycosylase (AGOG) in Thermococcus kodakarensis, an anaerobic, hyperthermophilic archaeon, and delineates its mechanism, kinetics, and substrate specificity. TkoAGOG is the major 8oxoG glycosylase in T. kodakarensis, but is non-essential. In addition to TkoAGOG, the major apurinic/apyrimidinic (AP) endonuclease (TkoEndoIV) required for archaeal base excision repair and cell viability was identified and characterized. Enzymes required for the archaeal oxidative damage base excision repair pathway were identified and the complete pathway was reconstituted. This study illustrates the conservation of oxidative damage repair across all Domains of life.
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ADN Glicosilasas/metabolismo , Reparación del ADN , Thermococcus/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Daño del ADN , ADN Glicosilasas/genética , Guanina/análogos & derivados , Guanina/metabolismo , Estrés Oxidativo , Thermococcus/genéticaRESUMEN
Genomic organization impacts accessibility and movement of information processing systems along DNA. DNA-bound proteins dynamically dictate gene expression and provide regulatory potential to tune transcription rates to match ever-changing environmental conditions. Archaeal genomes are typically small, circular, gene dense, and organized either by histone proteins that are homologous to their eukaryotic counterparts, or small basic proteins that function analogously to bacterial nucleoid proteins. We review here how archaeal genomes are organized and how such organization impacts archaeal gene expression, focusing on conserved DNA-binding proteins within the clade and the factors that are known to impact transcription initiation and elongation within protein-bound genomes.
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Archaea/genética , Archaea/metabolismo , Cromatina/metabolismo , Transcripción Genética , Proteínas Arqueales/metabolismo , ADN de Archaea/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
Control of electron flux is critical in both natural and bioengineered systems to maximize energy gains. Both small molecules and proteins shuttle high-energy, low-potential electrons liberated during catabolism through diverse metabolic landscapes. Ferredoxin (Fd) proteins-an abundant class of Fe-S-containing small proteins-are essential in many species for energy conservation and ATP production strategies. It remains difficult to model electron flow through complicated metabolisms and in systems in which multiple Fd proteins are present. The overlap of activity and/or limitations of electron flux through each Fd can limit physiology and metabolic engineering strategies. Here we establish the interplay, reactivity, and physiological role(s) of the three ferredoxin proteins in the model hyperthermophile Thermococcus kodakarensis We demonstrate that the three loci encoding known Fds are subject to distinct regulatory mechanisms and that specific Fds are utilized to shuttle electrons to separate respiratory and energy production complexes during different physiological states. The results obtained argue that unique physiological roles have been established for each Fd and that continued use of T. kodakarensis and related hydrogen-evolving species as bioengineering platforms must account for the distinct Fd partnerships that limit flux to desired electron acceptors. Extrapolating our results more broadly, the retention of multiple Fd isoforms in most species argues that specialized Fd partnerships are likely to influence electron flux throughout biology.IMPORTANCE High-energy electrons liberated during catabolic processes can be exploited for energy-conserving mechanisms. Maximal energy gains demand these valuable electrons be accurately shuttled from electron donor to appropriate electron acceptor. Proteinaceous electron carriers such as ferredoxins offer opportunities to exploit specific ferredoxin partnerships to ensure that electron flux to critical physiological pathways is aligned with maximal energy gains. Most species encode many ferredoxin isoforms, but very little is known about the role of individual ferredoxins in most systems. Our results detail that ferredoxin isoforms make largely unique and distinct protein interactions in vivo and that flux through one ferredoxin often cannot be recovered by flux through a different ferredoxin isoform. The results obtained more broadly suggest that ferredoxin isoforms throughout biological life have evolved not as generic electron shuttles, but rather serve as selective couriers of valuable low-potential electrons from select electron donors to desirable electron acceptors.
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Transporte de Electrón , Metabolismo Energético , Ferredoxinas/metabolismo , Thermococcus/enzimología , Thermococcus/metabolismo , Adenosina Trifosfato/metabolismo , Regulación de la Expresión Génica Arqueal , Thermococcus/genéticaRESUMEN
The sole unifying feature of Archaea is the use of isoprenoid-based glycerol lipid ethers to compose cellular membranes. The branched hydrocarbon tails of archaeal lipids are synthesized via the polymerization of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), but many questions still surround the pathway(s) that result in production of IPP and DMAPP in archaeal species. Isotopic-labeling strategies argue for multiple biological routes for production of mevalonate, but biochemical and bioinformatic studies support only a linear pathway for mevalonate production. Here, we use a combination of genetic and biochemical assays to detail the production of mevalonate in the model archaeon Thermococcus kodakarensis. We demonstrate that a single, linear pathway to mevalonate biosynthesis is essential and that alternative routes of mevalonate production, if present, are not biologically sufficient to support growth in the absence of the classical mevalonate pathway resulting in IPP production from acetyl-CoA. Archaeal species provide an ideal platform for production of high-value isoprenoids in large quantities, and the results obtained provide avenues to further increase the production of mevalonate to drive isoprenoid production in archaeal hosts.