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

Summary

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 T. 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 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.

Plain language summary:

Transcription elongation complexes that generate RNA transcripts from DNA templates must remain processively engaged for long periods and overcome obstacles in their path. Conserved transcription factors aid RNA polymerase in overcome translocation barriers.

Graphical Abstract

Introduction:

All eukaryotes and most archaea – to the exclusion of most Crenarchaeota – encode histone proteins to organize their genomes (Sandman and Reeve, 2000; Sandman and Reeve, 2001; Sandman and Reeve, 2006; Mattiroli et al., 2017; Bhattacharyya et al., 2018), and the resultant chromatin structures influence recognition of promoter elements (Nalabothula et al., 2013), alter transcription elongation rates (Chang and Luse, 1997; Xie and Reeve, 2004; Luse et al., 2011), change positions of transcription pausing, and can impact elongation-termination decisions (Gehring and Santangelo, 2015). Archaeal histone structures are nearly identical to the core structures of their eukaryotic counterparts, archaeal histone-DNA interactions align to the same nucleosome positioning code that was established for Eukarya (Segal et al., 2006; Nalabothula et al., 2013), and the three-dimensional structure of archaeal chromatin is nearly identical to the nucleosome (Soares et al., 2000; Mattiroli et al., 2017; Bhattacharyya et al., 2018). The superhelically-wrapped DNA shares the geometry, diameter, pitch, and writhe of the eukaryotic nucleosomal superhelix, and specific protein-DNA contacts that stabilize archaeal chromatin are conserved in eukaryotes (Luger et al., 1997; Mattiroli et al., 2017; Bhattacharyya et al., 2018). The eukaryotic nuclear RNA polymerases (RNAPs) and the archaeal RNAP thus regularly encounter – and must overcome – nearly identical histone-based barriers to transcription elongation (Kireeva et al., 2005; Teves et al., 2014; Mattiroli et al., 2017; Ehara et al., 2017; Kujirai et al., 2018).

Eukaryotic genomes encode many transcription factors that increase the processivity of RNAP, as well as histone remodeling and modification complexes that evict, reposition, and chemically alter histone-histone or histone-DNA interactions. Many of these complexes are required for proper growth and development, and the essential nature of so many complexes implies that the chromatin-imposed restrictions on transcription are a major force in dictating gene expression on a genome-wide scale and throughout temporal development. Given the structural and catalytic similarities between the archaeal and nuclear eukaryotic RNAPs (Werner and Grohmann, 2011; Jun et al., 2014; Murakami et al., 2015), most specifically RNA polymerase II (Pol II), and the identical histone-DNA contacts found in archaeal and eukaryotic chromatin, the global regulatory potential of chromatin is also imposed on Archaea (Luger et al., 1997; Sandman and Reeve, 2001; Marc et al., 2002; Reeve et al., 2004; Sandman et al., 2008a; Mattiroli et al., 2017; Bhattacharyya et al., 2018).

In contrast to Eukarya, archaeal histone-based chromatin is not known to be post-translationally regulated, and bioinformatic analyses of archaeal genomes have yet to reveal any obvious histone remodeling or histone modification machineries that are ubiquitous throughout eukaryotic genomes (Li et al., 2000; Koonin, 2015; Zaremba-Niedzwiedzka et al., 2017; Bhattacharyya et al., 2018). Binding of archaeal histones to transcription templates dramatically slows progression of the archaeal transcription apparatus in vitro, and changes to the chromatin landscape affect genome-wide expression in vivo (Xie and Reeve, 2004; Mattiroli et al., 2017).

Given the absence of conserved chromatin remodeling/modification machinery in Archaea, conserved transcription factors likely play the dominant role in aiding transcription through histone-bound DNAs (Werner and Grohmann, 2011). All archaea encode two conserved transcription factors – Spt5 (and its binding partner Spt4; Spt4-Spt5 is also commonly termed DSIF in metazoans (Wada et al., 1998; Bernecky et al., 2017; Vos et al., 2018)) and TFS (TFIIS in eukaryotes) – that are predicted to assist elongation through protein-bound templates (Werner and Grohmann, 2011). Any contributions of TFS or Spt4-Spt5 in aiding transcription through a chromatin barrier likely result from distinct mechanisms (Mattiroli et al., 2017). Spt5 (NusG in Bacteria) is the only universally conserved transcription factor (Werner and Grohmann, 2011). Spt5, often in complex with Spt4, is known to bind directly to RNAP and regulate the transition to stable elongation, promoter-proximal pausing, and processive elongation (Werner and Grohmann, 2011; Martinez-Rucobo et al., 2011; Guo et al., 2015; Bernecky et al., 2017; Ehara et al., 2017). Spt5 binds to RNAP via the clamp domain and facilitates increased processivity likely by retaining the clamp domain in a closed configuration (Grohmann et al., 2011; Schulz et al., 2016). Alignment of the template strand in the active center of RNAP is likely influenced by Spt4-Spt5 association with RNAP, and Spt4-Spt5 helps prevent arrest of Pol II via interactions with downstream DNA (Klein et al., 2011; Martinez-Rucobo et al., 2011; Grohmann et al., 2011; Schulz et al., 2016).

Bacterial RNAP, archaeal RNAP, and eukaryotic RNAP I/II/III all exhibit intrinsic endonuclease activity that can be stimulated by interactions with GreA and GreB (bacteria), TFS (archaea), TFIIS (also termed SII, eukarya – Pol II) (Hausner et al., 2000; Fish and Kane, 2002; Laptenko et al., 2003; Lange and Hausner, 2004; Miropolskaya et al., 2017). TFS and TFIIS are homologous, whereas GreA/GreB are analogous in function. All cleavage stimulatory factors bind RNAP near the secondary channel and extend protein fingers towards the active center of RNAP. The tips of these protein extensions typically contain and donate acidic residues to the RNAP active center that stimulate endonuclease activity (Lange and Hausner, 2004; Symersky et al., 2006; Miropolskaya et al., 2017; Fouqueau et al., 2017). TFS interacts with RNAP through the secondary channel (Symersky et al., 2006) and primarily acts to restore catalytic activity to transcription elongation complexes (TECs) that have paused and subsequently reverse translocated to position an internal phosphodiester linkage in the bipartite active center of RNAP (Park et al., 2002; Symersky et al., 2006; Klein et al., 2011; Sekine et al., 2015; Lisica et al., 2016; Xu et al., 2017). The lack of an RNA 3’ -OH group in the active site of RNAP prevents further transcription. Continued pausing of RNAP may lead to DNA replication complex/TEC collisions, resulting in genomic instabilities (Helmrich et al., 2013). Such ‘backtracked’ complexes are commonly the result of TECs encountering a strongly-bound protein or protein complex - such as histone-bound DNA – that slows/blocks RNAP forward translocation, thus stimulating TECs to reverse translocate. Backtracked TECs may resume productive elongation either by spontaneous isomerization back to the forward position, thereby reestablishing the 3’ end of the RNA in the active center, or by endonucleolytic cleavage of the RNA from a backtracked position that shortens the RNA and generates a new 3’ end in the active center of RNAP (Artsimovitch and Landick, 2000; Symersky et al., 2006; Ehara et al., 2017; Vos et al., 2018). 1-Dimensional diffusion forward to restore the RNA 3’ end to the active center is stochastic and limited to shallow backtracking events. Endonucleolytic cleavage of the RNA to provide the active site with a new 3’ end is independent of backtracking depth and is enhanced by protein factors, including TFS. Resumption of elongation permits RNAP to approach the downstream barrier again, and if the protein roadblock is retained, multiple rounds of backtracking, subsequent cleavage, and elongation may occur.

The purified transcription system from the histone-encoding euryarchaeon Thermococcus kodakarensis provides an ideal platform to investigate the roles of Spt4-Spt5 and TFS on transcription elongation on protein-free and histone-bound DNA templates (Xie and Reeve, 2004; Gehring and Santangelo, 2015; Walker and Santangelo, 2015; Gehring et al., 2016). We demonstrate first that sufficient histone proteins are present in vivo to fully saturate the archaeal genome, and that in vitro, addition of histone-proteins to DNA templates that contain stalled TECs results in the spontaneous assembly of chromatin structures that dramatically decrease ensemble transcription elongation rates. We then evaluated how different mechanisms of stabilizing TECs via binding of TFS, Spt4- or Spt5-alone, and Spt4-Spt5 complexes facilitated elongation through histone-bound DNAs and protein-free templates.

TFS stimulates endonucleolytic transcript cleavage and by reactivating backtracked TECs dramatically increases elongation rates on both protein-free and histone-bound DNA templates. A TFS variant that lacks cleavage-stimulatory activity was unable to accelerate transcription on protein-free or histone-bound DNAs. Addition of Spt4 or Spt5 alone had minimal effects on transcription elongation rates on protein-free or histone-bound templates and did not stimulate endonucleolytic cleavage of backtracked TECs nor reduce backtracking to any appreciable extent. Reconstitution of an Spt4-Spt5 complex did result in modest increases in the elongation rates on protein-free and histone-bound templates, suggesting that stabilizing TECs through clamp-closure can also assist in elongation through chromatin. The results obtained in vitro suggest that TFS is the primary factor that assists transcription elongation in vivo while Spt4-Spt5 provides an important secondary positive effect on transcription rate.

The essential nature of TFS, Spt4 and Spt5 in T. kodakarensis (Santangelo et al., 2008a; Hileman and Santangelo, 2012; Farkas et al., 2013), and the retention of each factor in almost all archaeal genomes (Werner and Grohmann, 2011), corroborates this inference and suggests that elongation through archaeal histone-bound DNA is opportunistic in nature. TECs repeatedly collide with protein barriers, backtrack and reattempt to transcribe through the barrier after regeneration of the RNA 3’ end within the active center of RNAP. TFS-stimulating endonucleolytic cleavage appears to be the most efficient mechanism to restore a catalytically active RNAP that can repetitively attempt to translocate through downstream protein-roadblocks. Spt4-Spt5 likely enhance elongation rates by binding to the clamp domain of RNAP, while making additional contacts with the downstream non-template strand stabilizing the TEC, facilitating processive elongation through chromatin by reducing pausing.

Results:

The genomes of T. kodakarensis are completely bound by histone proteins.

To ensure that in vitro studies on chromatin templates accurately reflected in vivo conditions we determined the concentration of histone proteins in T. kodakarensis. Quantitative Western blots, using DNaseI treated cellular lysates derived from T. kodakarensis cells and polyclonal antibodies that recognize both histone isoforms (HTkA and HTkB) revealed the steady-state abundance of histone proteins in vivo (Figure 1).

Figure 1. The genomes of T. kodakarensis are completely bound by histone proteins.

Known amounts of purified HTkA and HTkB were used as standards to generate quantitative linear regressions of Western-blot signal intensities for each histone variant. Western-blot signal intensities resultant from total histone-proteins present in aliquots from triplicate (A, B & C) lysates of T. kodakarensis cells were then used to extrapolate total histone-concentrations in vivo. The quantitative Western blot analyses of DNaseI treated T. kodakarensis lysates with polyclonal anti-HTkA antibodies demonstrates histone protein levels – HTkA and HTkB – are sufficient to bind the entirety of the the T. kodakarensis genomes (see M&M for details).

Establishing Western blot signal intensity curves using known concentrations of highly-purified HTkA and HTkB (Nalabothula et al., 2013) allowed us to establish the total number of histone molecules in cellular lysates (~2.5 ×10⁶ histone proteins per cell). T. kodakarensis is polyploid, retaining ~7–19 genomes per cell (Spaans et al., 2015), however, cells retain sufficient histone proteins to completely bind all genomes. Thus, histone proteins were added to in vitro transcription reactions in saturating amounts (>1 histone-dimer per 30 bp DNA).

Transcription Factor S (TFS), but not Spt4-Spt5, stimulates archaeal RNAP RNA-cleavage activity.

The component-purified, promoter-directed transcription system from T. kodakarensis permits formation of stalled TECs at defined template positions via nucleotide deprivation (Figure 2a). When conditions do not permit continued polymerization, TECs₊₅₈ spontaneously backtrack and slowly cleave nascent transcripts to generate a range of TECs with transcripts ranging from ~+50–58 (Figure 2b, lanes 7–11). When TECs₊₅₈ are provided with even low concentrations of ATP, GTP, and UTP, any TECs that backtrack and cleave their transcripts immediately resynthesize to +58 (Figure 2b, lanes 2–6). The position of TECs on such templates is thus dynamic, and addition of TFS dramatically stimulated transcript cleavage in backtracked TECs (Figure 2b, lanes 12–16). A TFS variant, wherein two conserved acidic residues were replaced with alanines (D90A, E91A; TFS^DE-AA), was unable to produce the same cleavage stimulatory effect as TFS^WT and even slightly impeded RNAP endonuclease activity (Figure 2b, lanes 17–21). The inability of TFS^DE-AA to properly donate acidic residues to the active site of RNAP abrogates its function as a cleavage stimulatory factor.

Figure 2. TFS, but not Spt4-Spt5, stimulates intrinsic RNAP endonuclease activity.

a) Biotinylated DNA templates permit promoter directed transcription to generate stalled TECs at the end of a 58 bp C-less cassette. Using nucleotide-deprivation, RNAPs positioned at +58 were isolated using paramagnetic streptavidin-coated beads. b) Upon incubation at 85°C, TECs₊₅₈ spontaneously backtrack and cleave nascent transcripts (lanes 7–11) to yield TECs_~+50–58. When NTPs (ATP, GTP, & UTP) are present, TECs rapidly re-elongate to +58 (lanes 2–6). The rate of nascent transcript cleavage is stimulated by addition of TFS^WT (lanes 12–16) but not by addition of TFS^DE-AA (lanes 17–21). Reaction aliquots were removed after 15, 30, 60, 120 and 420 seconds (left to right). c) Coomassie-stained, SDS-PAGE of purified TFS^WT and the inactive mutant TFS^DE-AA. Lane M contains size standards labeled in Kda to the left. d) TEC backtracking and nascent transcript cleavage is unaffected by the addition of Spt4, Spt5 or the Spt4-Spt5 complex. e) Coomassie-stained, SDS-PAGE of purified Spt4 and Spt5. Lane M contains size standards labeled in Kda to the left.

Backtracking can result from extended pausing (Nudler, 2012; Weixlbaumer et al., 2013; Imashimizu et al., 2013; Hein et al., 2014; Imashimizu et al., 2015; Lerner et al., 2016; James et al., 2016; Gabizon et al., 2018), and the configuration of mobile-domains of RNAP is known to modulate the propensity to pause and the duration of pausing (Martinez-Rucobo et al., 2011; Grohmann et al., 2011; Chakraborty et al., 2012; Weixlbaumer et al., 2013; Hein et al., 2014; Jun et al., 2014; Schulz et al., 2016; Sheppard et al., 2016; Feklistov et al., 2017; Kang et al., 2017; Bernecky et al., 2017; Duchi et al., 2018). We thus examined whether addition of Spt4 and/or Spt5 would influence the efficiency of RNA cleavage, with transcript cleavage also serving as a proxy for the propensity to, and depth of backtracking. In contrast to the stimulated RNA cleavage observed with TFS^WT, the addition of Spt4 and/or Spt5 did not influence RNA cleavage nor obviously affect the rate, depth, or propensity for backtracking (Figure 2d).

Archaeal transcription is impeded by histone-based chromatin.

Archaeal chromatin can be formed with a single histone protein and archaeal histones spontaneously bind DNA to assemble chromatin in vitro at the same positions and density as in vivo (Nalabothula et al., 2013; Mattiroli et al., 2017). These attributes, and the simplicity of the archaeal transcription apparatus, permitted us to first assemble washed, promoter-proximal, nucleotide-deprived, stalled TECs (one TEC per template) on DNA templates that could then be spontaneously bound by archaeal histones to assemble chromatin structures immediately downstream of the TECs. Histone-based chromatin structures were positioned by incorporating two, 60-bp tandemly repeated, SELEX-derived, optimized archaeal histone-positioning sequences (HPSs) (Bailey et al., 2000; Sandman et al., 2001) downstream of the stalled TECs (Figure 3a). The HPSs supported the spontaneous and consistent binding of histones that hindered MNase digestion and resulted in characteristic protection patterns derived from archaeal chromatin (Figure 3b) (Grayling et al., 1997). Histone-binding was monitored via electro-mobility shift assays (Figure 3c) (Marc et al., 2002; Sandman et al., 2008b), and at saturating histone concentrations (>1 histone dimer per 30 bp) (Mattiroli et al., 2017), the DNA templates were completely histone bound.

Figure 3. TFS increases the rate of elongation and full-length transcript production on protein-free and histone-bound templates.

a) TECs₊₅₈ can be generate on biotinylated C-less cassettes, washed, and then incubated with HTkB to generate well-positioned downstream histone barriers. b) Ethidium-bromide stained, agarose electrophoresis demonstrates that HTkB-binding protects DNAs from MNase digestion (lanes 9–13) under conditions where protein-free templates are rapidly degraded (lanes 3–7). Reactions aliquots were removed after 0, 1, 2, 3, 4, and 5 minutes. Lane M contains DNA size standards in base pairs labeled to the left. c) Native electrophoresis and ethidium bromide staining demonstrate that HTkB binding fully saturates and shifts the DNA templates. d) On both protein-free (lanes 2–16) and histone-bound templates (lanes 17–31), the addition of TFS^WT but not TFS^DE-AA accelerates ensemble elongation rates and stimulates production of full-length +230 nt transcripts. Reactions aliquots were removed after 15, 30, 60, 120, and 300 seconds. e) Quantification of full-length transcript levels (n ≥ 3) in the absence and presence of TFS on protein-free and histone-bound templates demonstrates that TFS accelerates transcription and increases full-length transcript yields. The amount of full-length transcripts at 5 minutes on protein-free templates in the absence of TFS is set to 1.0.

By first generating and isolating TECs₊₅₈ on templates that were homogenously histone-bound or remain protein-free, the rates of elongation on each template upon NTP addition were determined (Figure 3d). Transcription rapidly restarts regardless of the presence/absence of downstream protein-barriers and the population of TECs quickly becomes nonsynchronous; template positions that hinder elongation and direct pausing are evident on protein-free templates, but the bulk of TECs reach the end of the template to generate +230 nt transcripts without substantial delay.

Addition of either histone from T. kodakarensis (Histone A = TK1413, HTkA; Histone B = TK2289, HTkB) (Fukui et al., 2005; Čuboňováa et al., 2012; Nalabothula et al., 2013; Mattiroli et al., 2017) results in dramatically altered elongation patterns and rates of transcription. The overall elongation rate and resultant pace of RNA synthesis from archaea TECs are dramatically reduced when DNA is bound by archaeal histones. Compared to transcription on protein-free templates, chromatin blocked ~80% of TECs from generating full-length transcripts, and the blocked TECs often arrested or paused for extended periods at multiple positions. The inhibition of transcription elongation observed for archaeal components almost exactly matches the impediment observed of Pol II transcribing through a well-positioned nucleosome (Xie and Reeve, 2004; Kireeva et al., 2005; Kim et al., 2010; Luse et al., 2011; Bintu et al., 2012; Nock et al., 2012; Kwak and Lis, 2013; Ishibashi et al., 2014; Studitsky et al., 2016; Lee and Blobel, 2016; Xu et al., 2017; Crickard et al., 2017; Van Oss et al., 2017; Kujirai et al., 2018). The initial collision results in the greatest obstacle, and when the TEC escapes this initial collision, transcription pauses every ~10–15 bp while the TEC traverses the histone complex. The duration of the initial pause is much greater than subsequent pauses, implying that the rate limiting step to transcription on chromatin templates – in both archaea and eukaryotes – is disrupting the first set of conserved histone-DNA contacts.

TFS increases the rate of elongation and full-length transcript production on protein-free and histone-bound templates by reactivating backtracked complexes.

To assess the role TFS and its cleavage-stimulatory activity on the rate and efficiency of transcription though chromatin, purified TFS^WT and TFS^DE-AA were added at the time of transcription restart on protein-free and histone-bound templates. On templates lacking chromatin, TFS^WT, but not TFS^DE-AA increased the rate and total amount of full-length transcript production (Figure 3d, lanes 7–11 & 8–12, respectively). Addition of TFS^WT reproducibly resulted in nearly-twice the total number of full-length transcripts, and these additional full-length transcripts were generated from TECs that were paused/arrested when TFS was not present or when TFS^DE-AA was added. The addition of TFS^WT to TECs elongating through chromatin dramatically increased both the percentage of full-length transcripts and the rate of full-length transcript production from histone-bound templates. Just ~20% of the full-length transcripts generated on protein-free templates were observed on chromatin templates in the absence of TFS, and the rate of full-length transcript production was reduced by ~20–25-fold. Addition of TFS^WT restored ~90% of full-length transcript production and increased the ensemble rate of elongation through chromatin ~4-fold (Figure 3e). As observed on protein-free DNA, addition of TFS^DE-AA to TECs transcribing chromatin had no discernable effects, demonstrating that the transcript-cleavage-stimulatory function of TFS is critical for accelerating transcription on histone-bound DNA. The stimulatory activities of TFS^WT on protein-free and histone-bound DNA suggest that RNAP often pauses - in response to DNA sequence motifs and proteinaceous-roadblocks – long enough to backtrack. These backtracking events hinder elongation and demonstrate that cleavage and synthesis cycles more rapidly recover active TECs than spontaneous isomerization to the restore the RNA 3’ end in the active center of RNAP.

Spt5 and Spt4 together, but not individually, facilitate elongation through chromatin.

Spt4-Spt5 binds directly to RNAP and stabilizes a closed-clamp configuration that facilitates elongation and TEC stability (Wada et al., 1998; Martinez-Rucobo et al., 2011; Grohmann et al., 2011; Guo et al., 2015; Bernecky et al., 2017; Ehara et al., 2017; Kang et al., 2018; Vos et al., 2018). As pausing and subsequent backtracking can be influenced by inter-domain movements of RNAP (Martinez-Rucobo et al., 2011; Chakraborty et al., 2012; Weixlbaumer et al., 2013; Hein et al., 2014; Jun et al., 2014; Blombach et al., 2016; Feklistov et al., 2017; Kang et al., 2017; Duchi et al., 2018), we sought to determine whether Spt4-Spt5 binding to RNAP would reduce pausing or accelerate transcription on protein-free and histone-bound templates. Addition of either Spt4 or Spt5 alone (Figure 4a, lanes 7–11 & lanes 12–16, respectively; Figure 4b) did not alter the ensemble rate of elongation nor the total production of full-length transcripts on protein-free templates. When added together, the Spt4-Spt5 complex (Figure 4a, lanes 17–21; Figure 4b) resulted in a modest (~20%) increase in total transcript production and a reproducible, but small increase in elongation rate (Hirtreiter et al., 2010).

Figure 4. The Spt4-Spt5 complex, but neither Spt4 or Spt5 alone, increases the rate of elongation and full-length transcript production on protein-free and histone-bound templates.

a) TECs₊₅₈ were assembled, washed, and HTkB then added (lanes 23–42) or left out (lanes 2–21) before elongation restart in the presence or absence of Spt4 and/or Spt5. Only the Spt4-Spt5 complex accelerates ensemble elongation rates and stimulates production of full-length +230 nt transcripts. b) Quantification of full-length transcript levels (n ≥ 3) in the absence and presence of Spt4 and/or Spt5 on protein-free and histone-bound templates demonstrates that the Spt4-Spt5 complex accelerates transcription and increases full-length transcript yields. The amount of full-length transcripts at 2 minutes on protein-free templates in the absence of either factor is set to 1.0.

A ~40% increase in total full-length transcript production and a modest (~1.5-fold) acceleration of elongation rate were obtained when the Spt4-Spt5 complex was added to TECs transcribing chromatin, but neither factor alone resulted in increased elongation rates or proficiency on histone-bound DNA [Spt4 (Figure 4a, lanes 29–33) or Spt5 (Figure 4a, lanes 34–38)]. Thus, although not as robust as the improvements observed based on addition of TFS^WT, the conserved Spt4-Spt5 complex does aid in transcription elongation rates and efficiencies on both protein-free (Hirtreiter et al., 2010) and histone-bound DNAs (Figure 4b).

TFS, Spt4 and Spt5 are essential for growth of T. kodakarensis.

T. kodakarensis has an attractive genetic system that permits rapid construction of strains with genomic modifications (Santangelo et al., 2008a; Hileman and Santangelo, 2012; Farkas et al., 2013; Jäger et al., 2014; Gehring et al., 2017). Hundreds of genes have successfully been deleted/modified using an integration-excision based homologous recombination system (Sato et al., 2005; Santangelo et al., 2010; Hileman and Santangelo, 2012; Gehring et al., 2017). Two excision events are possible, but only one event results in the modification or deletion of the target locus, and by analyzing the results of hundreds of individual excision events, a statistical definition of essentiality can be derived (Hileman and Santangelo, 2012). For most non-essential genes, the expected 1:1 ratio of excision events that result in restoration of the original genome versus deletion/modification of the target locus have been observed (Gehring et al., 2017). Using established techniques (Gehring et al., 2017), we exhaustively attempted to delete TK0533 (encoding TFS), Spt4 (TK1698) and Spt5 (TK1419) from the T. kodakarensis genome. Despite analyzing >200 individual excision events for each locus, no strains were recovered with the desired targeted deletions. These results imply that that these well-conserved elongation factors are necessary for proper gene expression in vivo and that neither elongation factor (TFS and Spt4-Spt5) alone can assist RNAP through all obstacles encountered during transcription elongation. The modest effect of Spt4-Spt5 suggests that a yet undiscovered primary function for this conserved complex likely exists, possibly relating to the coupling of transcription to translation (French et al., 2007) or termination when transcription becomes uncoupled from translation (Santangelo et al., 2008b).

Discussion

The activities of all macromolecular complexes that interact with DNA must access DNA in a nucleoid- or chromatin-context, and for processive assemblies – such as replication or transcription complexes – the obstacles presented by DNA-bound proteins can dramatically alter elongation rates. Eukaryotic- and most archaeal-genomes are bound by histone proteins (Mattiroli et al., 2017), and in T. kodakarensis, histone protein concentrations are sufficiently high to bind the entirety of the genomes (Spaans et al., 2015). The replicative MCM helicase is sufficient powerful enough to easily unwind DNA bound by archaeal histones (Shin et al., 2007). In contrast, the rate of transcription on archaeal histone-bound DNAs is dramatically slowed compared to protein-free transcription elongation (Xie and Reeve, 2004).

While eukaryotic genomes encode a wealth of factors to both perturb and strengthen the interactions of histones with DNA, the main barrier to the expression of genes is the core histone fold and its interactions with DNA (Chang and Luse, 1997; Kireeva et al., 2005; Kim et al., 2010; Luse et al., 2011; Bintu et al., 2012; Nock et al., 2012; Kwak and Lis, 2013; Studitsky et al., 2016; Lee and Blobel, 2016; Van Oss et al., 2017; Kujirai et al., 2018). No histone modification machinery has been identified in Archaea, nor has evidence of post-translational modification of archaeal histones emerged (Peeters et al., 2015). The ability of archaeal histones to form hetero- or homo-dimeric pairings (Reeve et al., 2004) suggests there may be a yet undiscovered method of regulation in the third domain. Our ability to assemble transcription elongation complexes with or without histones provides a quantitative assay to characterize potential novel and conserved archaeal factors that may accelerate transcription through histone-bound DNA.

Archaeal genomes typically retain only two known and conserved transcription factors (Werner and Grohmann, 2011) – TFS and Spt4-Spt5 – which are known to function through different mechanisms to promote transcription elongation. We demonstrate that addition of TFS stimulates the endonucleolytic cleavage activity of RNAP, and that such stimulation is critical to accelerate transcription elongation through the normally very restrictive barrier presented by assemblages of archaeal histones bound to DNA (Xie and Reeve, 2004). Addition of TFS triples the ensemble elongation rate and nearly completely restores production of full-length transcripts on histone-bound templates. The ability of TFS to stimulate cleavage of nascent transcripts is critical for such stimulation, as a TFS variant that does not contribute acidic residues to the RNAP active site does not alter elongation kinetics or full-length transcript production (Kettenberger et al., 2004; Schweikhard et al., 2014). Spt4-Spt5, when combined, also modestly increase the rate of elongation and promote production of full-length transcripts on both protein-free and histone-bound DNAs (Hirtreiter et al., 2010). Neither Spt4 nor Spt5 alone influences RNA cleavage, and only together can Spt4-Spt5 stabilize RNAP such that intrinsic and histone-induced pausing is reduced.

We present a model of transcription elongation through chromatin templates that incorporates the known activities of TFS and Spt4-Spt5 to explain the mechanistic features that facilitate transcription elongation through a chromatin barrier (Figure 5). Our experimental results support a model wherein repeated rounds of backtracking, cleavage, and elongation permit TECs to continually approach the histone-imposed barrier leading to opportunistic traversal of the chromatin barrier. When the chromatin barrier is sufficiently dynamic, the TEC – in complex with TFS and Spt4-Spt5 – can displace histone proteins and continue elongation to the end of the template (Figure 5, panels I-IV). Uninterrupted elongation is uncommon, and most TECs pause (Figure 5, panel Ib) upon encountering a stable downstream histone-barrier. Extended pausing results in backtracking (Artsimovitch and Landick, 2000) (Figure 5, panel Ic), and the depth of backtracking may be limited by any upstream chromatin landscape. TFS-stimulated cleavage of the nascent transcript (Figure 5, panel Id) reestablishes a 3’ OH in the RNAP active center (Lange and Hausner, 2004). Due to the spontaneous nature of archaeal histone interactions with DNA, both upstream and downstream segments of DNA are histone-bound. Most TECs will go through multiple rounds of elongation, pausing, backtracking, cleavage and synthesis to traverse a chromatin template. The activities of the Spt4-Spt5 complex decrease pausing likely by stabilizing a closed clamp configuration, limiting jaw and clamp movements, and through interactions with the non-template strand (Klein et al., 2011; Grohmann et al., 2011; Guo et al., 2015; Schulz et al., 2016; Crickard et al., 2017; Bernecky et al., 2017). The totality of these interactions increases RNAP processivity through the remaining barrier while not interfering with the backtracking rescue activity of TFS.

Figure 5. Model for transcription-factor aided elongation through archaeal histone-based chromatin.

Ia) TFS and Spt4-Spt5 are associated with RNAP, while the downstream histone-based chromatin landscape is dynamic. Ib) Collision with a downstream histone-DNA barrier results in RNAP pausing. Ic) Extended pausing results in RNAP backtracking and movement of the RNA 3’-end from the active center to the secondary channel. Id) TFS stimulated endonucleolytic transcript cleavage by RNAP generates a new 3’ OH in the RNAP active site. II) In repeated rounds of synthesis and cleavage the histone-based chromatin landscape has shifted allowing further progression by RNAP. III) Spt4-Spt5 likely reduce pausing while traversing histone-bound DNA by stabilizing the closed-clamp configuration that may stimulate forward translocation. IV) The combinatorial activities of TFS and Spt4-Spt5 allow RNAP to transcribe the full-length of a DNA template through a shifting histone landscape.

Our results demonstrate the importance of the universally conserved protein Spt5 and the conserved activity of TFS in modulating various aspects of RNAP activity to overcome both sequence-induced pauses and histone-induced barriers. The ability of TFS (or Gre factors in Bacteria) (Toulmé et al., 2000; Miropolskaya et al., 2017) to restore RNAP from a backtracked position, and Spt4-Spt5 interactions to maintain a closed-clamp, processive confirmation of RNAP likely stem from LUCA and remain in all extant organisms to facilitate transcription elongation through protein-based roadblocks. The evolution of histone-tails and extensions, and post-translational histone-modifications likely further impacts transcription elongation and it is plausible that these eukaryotic chromatin modifications necessitated the evolution of eukaryotic chromatin-remodeling complexes.

Experimental Procedures

Protein Purifications.

RNAP, TBP, TFB, HTkA and HTkB were purified as described previously(Santangelo et al., 2007; Nalabothula et al., 2013). Spt4, Spt5, TFS^WT, and TFS^DE-AA were purified from Rosetta2 E. coli cells (Millipore Sigma) containing modified pQE-80L expression vectors (Qiagen) containing His₆-Spt4-, Spt5-, TFS^WT- or TFS^DE-AA encoding sequences, respectively, grown in LB medium with 30 μg/mL chloramphenicol and 100 μg/mL ampicillin. Expression of each protein was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside, and cultures were grown for an additional 3 h at 37°C with shaking (~225 rpm). Biomass was harvested, resuspended and lysed via sonication (3 mL/g of pellet) in lysis buffer (25 mM Tris-HCl pH 8.0, 50 mM NaCl). Cellular lysates were clarified by centrifugation, (~20,000 x g, 20 min, 4°C). TFS proteins were partially purified by heating the clarified cell lysates to 85° C for 30 min, followed by passage and fractionation of cleared supernatants through a cellulose phosphate column. The column was equilibrated in 25 mM Tris-HCl pH 8.0, 50 mM NaCl, and resolved with a linear gradient of 50 mM – 1 M NaCl in 25 mM Tris-HCl pH 8.0. Fractions containing TFS were identified by SDS-PAGE, pooled, dialyzed into storage buffer (25mM Tris-HCl pH 8.0, 100mM KCl, 10mM β-ME, and 50% Glycerol) and quantified using a Bradford Assay(Bradford, 1976). His₆-Spt4 was partially purified by heating clarified cell lysate at 85° C for 30 min, followed by passage and fractionation of the cleared supernatant through a 1 mL Hi-TRAP chelating column (GE Healthcare) preequilibrated with NiSO₄. The column was washed in 25 mM Tris-HCl pH 8.0, 500 mM NaCl, 10 mM imidazole, and 10% glycerol and resolved with a linear imidazole gradient to 60% 25 mM Tris-HCl pH 8.0, 500 mM imidazole, 100 mM NaCl and 10% glycerol. Spt4 containing fractions were identified by SDS-PAGE, pooled, and dialyzed into storage buffer (25 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM β-ME, and 50% glycerol). Spt5 was partially purified by heating clarified cell lysate at 85° C for 30 min, followed by passage and fractionation of cleared supernatant through a S-100 size exclusion column (GE Healthcare) equilibrated in 25 mM Tris-HCl pH 7.4, 200 mM NaCl. Spt5 containing fractions were identified by SDS-PAGE, pooled, and dialyzed into storage buffer (25 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM β-ME, and 50% glycerol).

DNA template construction.

Double-stranded biotinylated DNA templates used in transcription reactions were PCR amplified from plasmids and gel purified as described(Walker et al., 2017).

Western Blot Analysis and Histone Quantification.

HTkA was used as an antigen to prepare polyclonal antibodies in rabbits (Cocalico Biologicals). Known amounts of HTkA and HTkB were loaded into gels as comparative quantification standards in adjacent lanes to DNaseI-treated clarified cell lysates (Nalabothula et al., 2013). Proteins were separated via SDS-PAGE, transferred to PVDF membranes, and probed with primary anti-HTkA antibodies. Addition of an IgG-HRP conjugated anti-rabbit secondary antibody allowed for detection by chemiluminescent ECL western blotting substrate (Thermo Fisher Scientific). The anti-HTkA polyclonal antibodies recognized both HTkA and HTkB with similar affinities, permitting a linear regression of HTkA and HTkB signal intensity to HTkA or HTkB amount in ng to be generated. Steady-state histone levels present T. kodakarensis lysates were sufficient to coat >85% percent of the genomes (assuming 30 bp/histone-dimer) even when 19 genomes were assumed per cell. 19 genomes (~2.08 Mbp each) represents ~40 Mbp of genomic DNA, or ~1.3 ×10⁶, thirty-bp histone-dimer binding positions. The total number of histone proteins – HTkA plus HTkB – was ~1.6 – 3.3 ×10⁶ per cell; ~2.5 ×10⁶ histone proteins - ~1.25 million histone dimers – on average; the ratio of HTkA to HTkB is unknown. Given that T. kodakarensis carries between ~7–19 genomes (13 genomes per cell on average) (Spaans et al., 2015), sufficient histone-dimers are normally present to bind the entirety of the T. kodakarensis genomes.

MNase assays.

Micrococcal nuclease digestions were performed as described (Mattiroli et al., 2017).

Histone-DNA binding assays.

Electromobility shift assays were performed as described (Bailey et al., 2000).

In vitro transcription reactions.

Assembly of preinitiation complexes (PICs) and elongation via NTP deprivation was carried out as described previously (Santangelo et al., 2007; Walker et al., 2017). To obtain stalled TEC₊₅₈, PICs were incubated with 200 μM ATP, 200 μM GTP, 10 μM UTP and 10 μCi [α−³²P]-UTP for 3 min at 85°C, then chilled to 4°C and biotinylated templates were captured with streptavidin coated paramagnetic particles (Promega). TECs₊₅₈ were thrice washed in 100 μl WB (20 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 M KCl) then resuspended in 10 mM Tris-HCl pH 8.0, 125 mM KCl, 5 mM MgCl₂, 1 mM DTT, and ± 10 μM ATP/GTP/UTP. For reactions on protein-free templates, aliquots of washed TECs were combined with equal volume reactions containing 10 mM Tris-HCl pH 8.0, 1 mM MgCl₂, 120 mM KCl, 8 mM DTT, ± Spt4, ± Spt5 or ± Spt4-Spt5, -or- ± TFS^WT or ± TFS^DE-AA. To ensure sufficient saturation of TECs, TFS was added at ~9 μM, whereas Spt4 and/or Spt5 were added at ~6 μM each. Elongation was reinitiated at 85°C by the addition of 25 μM NTPs and reaction aliquots were removed over a time course into 5 volumes of 1.2X STOP buffer (0.6 M Tris-HCl pH 8.0, 12 mM EDTA). Radiolabeled transcripts were recovered by addition of 7 μg of tRNA (total), equal volume phenol/chloroform/isoamyl alcohol (25:24:1, by volumes) extractions, and precipitations of the aqueous phase with 2.6 volumes 100% ethanol (Santangelo et al., 2007). Precipitated transcripts were resuspended in 95% formamide, 1X TBE, heated to 99°C for 5 minutes, rapidly chilled on ice, loaded and resolved in 10–20% polyacrylamide, 8M urea, 1X TBE denaturing gels. Radiolabeled RNA was detected using phosphorimaging (GE Healthcare). Gel images were analyzed using GE Imagequant 5.2 software.

To obtain TECs on histone-bound templates, TEC₊₅₈ were generated and captured as described above. TEC₊₅₈ were then resuspended in 20 mM Tris-HCl pH 8.0, 100 mM KCl, 4 mM MgCl₂, 3 mM DTT, 10 μM each of ATP, GTP, and UTP, and saturating amounts of HTkB for 30 min at 4°C; HTkB was added to 1.5 dimers per 30 bp of template DNA. Reactions were incubated at 85°C for 2 minutes prior to transcription restart by addition of 25 μM NTPs (for Figure 3) or 100 μM NTPs (for Figure 4) ± Spt4, ± Spt5 or ± Spt4-Spt5, -or- ± TFS^WT or ± TFS^DE-AA. Reactions were processed as above.