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

Abstract

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.

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).

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.

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.

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.

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.