Manipulating Archaeal Systems to Permit Analyses of Transcription Elongation-Termination Decisions In Vitro

Gehring, Alexandra M.; Santangelo, Thomas J.

doi:10.1007/978-1-4939-2392-2_15

Cited by 11 publications

(22 citation statements)

References 48 publications

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“…T. kodakarensis cultures were grown at 85°C under anaerobic conditions in artificial seawater medium supplemented with yeast extract, tryptone, and elemental sulfur (ASW-YT-S 0 ), as previously described (28,29,32). Culture growth was monitored by optical density measurements at 600 nm.…”

Section: Methodsmentioning

confidence: 99%

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

et al. 2017

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Many aspects of and factors required for DNA replication are conserved across all three domains of life, but there are some significant differences surrounding lagging-strand synthesis. In Archaea, a 5=-to-3= exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome specifically through interactions with GINS was identified and designated GAN (for GINS-associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation, and as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, and GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted, with no discernible effects on viability and growth. However, deletion of both Fen1 and GAN was not possible, consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo. RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1, but it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for the activation or stability of the archaeal MCM replicative helicase. IMPORTANCEThe mechanisms used to remove primer sequences from Okazaki fragments during lagging-strand DNA replication differ in the biological domains. Bacteria use the exonuclease activity of DNA polymerase I, whereas eukaryotes and archaea encode a flap endonuclease (Fen1) that cleaves displaced primer sequences. RNase HII and the GINS-associated exonuclease GAN have also been hypothesized to assist in primer removal in Archaea. Here we demonstrate that in Thermococcus kodakarensis, either Fen1 or GAN activity is sufficient for viability. Furthermore, GAN can support growth in the absence of both Fen1 and RNase HII, but Fen1 and RNase HII are required for viability in the absence of GAN.KEYWORDS Archaea, CMG complex, DNA replication, GINS-associated nuclease GAN, Okazaki fragment, RNase HII, flap endonuclease Fen1 D NA replication is catalyzed by a protein complex, designated the replisome, with structural and catalytic components tightly regulated, intertwined, and coordinated to facilitate both leading-and lagging-strand synthesis. The replisome composition is

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Section: Methodsmentioning

confidence: 99%

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

et al. 2017

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“…Our purified in vitro transcription system from T. kodakarensis permits promoter-directed transcription initiation on DNA templates attached to a solid-support, allowing stable TECs to be generated at defined template positions by nucleotide deprivation (Figure 3.1A) (56,61). TECs are resistant to repeated washing and 32 P-labeled nascent transcripts are detected in the bound or pellet fraction.…”

Section: Identification Of a Euryarchaeal Transcription Termination Amentioning

confidence: 99%

“…Santangelo TJ, Luyties O, and I contributed in data collection and analysis. We established a biochemical assay, using a robust in vitro transcription system dependent on purified RNAP and basal initiation factors from the model hyperthermophilic archaea Thermococcus kodakarensis to purify the first archaeal-encoded activity that can disrupt the TEC (38,56). Our assay is dependent on the disruption of stalled archaeal TECs that are normally extremely stable and remain intact even when challenged with the strong replicative minichromosome maintenance (MCM) helicase (57).…”

Section: Protein Purification and Co-isolation Of Tfe With Rnapmentioning

confidence: 99%

“…Several studies have identified critical interactions between TFE and the preinitiation complex that have furthered our understanding of TFE function during initiation (2,18,54). TFEα consists of two domains: a winged helix (WH) domain and a zinc ribbon domain (55,56); TFEβ contains a conserved WH domain and an FeS domain (20). The WH domain of TFEα contacts the upstream, nontemplate strand of DNA and helps form the open promoter complex through an unknown mechanism (18,46).…”

Section: Regulation Of Transcription Initiationmentioning

confidence: 99%

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Factor-dependent archaeal transcription termination

Walker

Luyties

Santangelo

2017

Proc. Natl. Acad. Sci. U.S.A.

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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.While many studies in archaea have focused on elucidating the mechanism of transcription initiation and elongation, studies on termination were slower to emerge.Transcription factors promoting initiation and elongation have been characterized in each Domain but transcription termination factors have only been identified in bacteria and eukarya.Here we characterize the first archaeal termination factor (termed Eta) capable of disrupting the transcription elongation complex, detail the rate of and requirements for Eta-mediated transcription termination and describe a role for Eta in transcription termination in vivo. Etamediated transcription termination is energy-dependent, requires upstream DNA sequences and disrupts transcription elongation complexes to release the nascent RNA to solution. Deletion of TK0566 (encoding Eta) is possible, but results in slow growth and renders cells sensitive to iii DNA damaging agents. Structure-function studies reveal that the N-terminal domain of Eta is not necessary for Eta-mediated termination in vitro, but Thermococcus kodakarensis cells lacking the N-terminal domain exhibit slow growth compared to parental strains. We report the first crystal structure of Eta that will undoubtedly lead to further structure-function analyses. The results obtained argue that the mechanisms employed by termination factors in archaea, eukarya, and bacteria to disrupt the transcription elongation complex may be conserved and that Eta stimulates release of stalled or arrested transcription elongation complexes.iv ACKNOWLEDGEMENTS

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“…Some differences in promoter sequence preferences and protein pairing have been noted in TBP-TFB isoform pairs (36)(37)(38)(39)(40)(41), but these minor differences are not on par with the clear but not always radical promoter sequence differences noted for alternative factors in bacterial transcription (39,42). TFE also appears essential, and it is currently unclear if this essentiality is due to necessary activities during transcription initiation or some other role in the transcription cycle (26,43,44).…”

Section: Basal Transcription Factorsmentioning

confidence: 99%

Transcription Regulation in Archaea

2016

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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. RNA polymerase (RNAP) is a well-conserved, multisubunit essential enzyme that transcribes DNA to generate RNA in all cells. Although RNA synthesis is carried out by RNAP, the activities of RNAP during each phase of transcription are subject to basal and regulatory transcription factors. Substantial differences in transcription regulatory strategies exist in the three domains (Bacteria, Archaea, and Eukarya). Only a single transcription factor (NusG or Spt5) is universally conserved (1, 2), and the roles of many archaeon-encoded factors have not been evaluated using in vivo and in vitro techniques. Archaea are reliant on a transcription apparatus that is homologous to the eukaryotic transcription machinery; similarities include additional RNAP subunits that form a discrete subdomain of RNAP (3, 4) as well as basal transcription factors that direct transcription initiation and elongation (5-8).The shared homology of archaeal-eukaryotic transcription components aligns with the shared ancestry of Archaea and Eukarya, and this homology often is exclusive of Bacteria. Archaea are prokaryotic, but the transcription apparatus of Bacteria differs significantly from that of Archaea and Eukarya.The archaeal transcription apparatus is most commonly summarized as a simplified version of the eukaryotic machinery. In some respects this is true, as homologs of only a few eukaryotic transcription factors are encoded in archaeal genomes, and archaeal transcription in vitro can be supported by just a few transcription factors. However, much regulatory activity in eukaryotes is devoted to posttranslational modifications of chromatin, RNAP, and transcription factors, and this complexity seemingly does not transfer to the Archaea, where few posttranslational modifications or chromatin-imposed regulation events are currently known. The ostensible simplicity of archaeal transcription is under constant revision, as more detailed examinations of archaeon-encoded factors become possible through increasingly sophisticated in vivo and in vitro techniques. This review will highlight the current understanding of archaeal transcriptio...

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Manipulating Archaeal Systems to Permit Analyses of Transcription Elongation-Termination Decisions In Vitro

Cited by 11 publications

References 48 publications

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

The GAN Exonuclease or the Flap Endonuclease Fen1 and RNase HII Are Necessary for Viability of Thermococcus kodakarensis

Factor-dependent archaeal transcription termination

Transcription Regulation in Archaea

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