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FttA is a CPSF73 homologue that terminates transcription in Archaea

Nature Microbiology volume 5, pages 545–553 (2020)Cite this article

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Abstract

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 genomes1,2,3 in which continued transcription would necessarily transcribe immediately adjacent genes and result in conflicts between the transcription and replication apparatuses4,5,6; 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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Fig. 1: FttA is a bona fide archaeal transcription termination factor.
Fig. 2: FttA-mediated termination shares mechanistic requirements of Rho-mediated bacterial transcription termination.
Fig. 3: FttA-mediated transcription termination is competitive with transcription elongation.
Fig. 4: Inhibition of FttA activity abolishes transcription termination in vitro and reduced FttA expression or activity alters steady-state RNA 3′ termini in vivo.

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Data availability

The data that support the findings of this study are available from the authors on reasonable request. Source data for Figs. 1e and 4h, as well as Extended Data Figs. 2a, 3d and 5 are included in this article and its Supplementary Information files.

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Acknowledgements

We thank members of the Santangelo laboratory for critical review of the manuscript. This work was supported by the National Institutes of Health grant no. GM100329 (to T.J. Santangelo).

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  1. Julie E. Walker

    Present address: Watchmaker Genomics, Boulder, CO, USA

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  1. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA

    Travis J. Sanders, Breanna R. Wenck, Jocelyn N. Selan, Mathew P. Barker, Stavros A. Trimmer, Julie E. Walker & Thomas J. Santangelo

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Contributions

T.J. Sanders performed in vitro transcription and RNase digestions, designed and purified FttA variants, designed and assisted in the construction of T. kodakarensis strain IR5, purified proteins and templates, generated qRT–PCR data, analysed data, built structural models, performed western blots, prepared figures and helped write the manuscript. B.R.W. performed in vitro transcription and RNase digestions, designed templates and FttA variants, manipulated TK1428 genomic sequences and prepared figures. J.N.S. generated FttA variants, purified transcription proteins, performed bioinformatic analyses, manipulated TK1428 genomic sequences to generate strain TK1428D, built structural models and prepared figures. M.P.B. and S.A.T. manipulated TK1428 genomic sequences, generated and analysed qRT–PCR data and assisted with western blots and protein purifications. J.E.W. purified FttA variants and performed in vitro transcription. T.J. Santangelo conceived and directed the project, wrote the manuscript, analysed data and prepared figures.

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Correspondence to Thomas J. Santangelo.

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Extended data

Extended Data Fig. 1 Transcription termination mechanisms commonly employed in Bacteria, Eukarya and Archaea.

Intrinsic transcription termination in Bacteria (a), Archaea (b), and for eukaryotic Pol III (c) results in release of the entire 5′-triphosphate-containing RNA transcript following transcription through a region of dyad-symmetry encoding an RNA hairpin immediately proceeded a T-rich non-template strand sequence (Bacteria)64 or T-rich non-template strand sequences (Archaea and eukaryotic Pol III)20,32,65,66,67,68,69. d, g, Factor-mediated bacterial transcription termination64, driven by rho or Mfd, also directs release of the entire nascent transcript and results in collapse of the TEC and recycling of RNAP. Rho-mediated termination is aided by NusG (Spt5 in Eukarya and Archaea)34,36,70,71,72. e, Release of the majority of the nascent transcript cannot be considered a bona fide termination event in-of-itself. RNA processing events, such as the endonucleolytic cleavage of the nascent RNA within eukaryotic Pol II TECs by the cleavage and polyadenylation factor complex (CPSF)16,25,50,57,61 yield a 5′-fragment that is often further processed – typically by the addition of a 3′-polyA tail for many Pol II transcripts – but also a 3′-fragment that is encapsulated within a still-stable TEC;28,60 the combined activities of CPSF and Xrn2 are necessary for normal termination patterns in Eukarya59,60,73. f, FttA can cleave the nascent transcript and terminate the archaeal transcription apparatus. g, h, Both bacterial Mfd and archaeal Eta can disrupt stalled TECs and release full-length transcripts by rewinding the transcription bubble.

Extended Data Fig. 2 FttA is highly conserved and shares structural and sequence homology with eukaryotic CPSF73.

Recombinant FttA (the protein product of T. kodakarensis TK1428; TK1428p) is 73.5 Kda, 85˚C thermotolerant and free of contaminating proteins. Lane M contains molecular weight standards in Kda. Data shown are from a single experiment. b, The crystal structure of FttA from Pyrococcus horikoshii (PDB: 3AF5)17 shown in chainbow coloring (N-terminus in purple to C-terminus in red) reveals two N-terminal KH-domains attached by a linker to a C-terminal MBL fold. Alpha-carbons of highly conserved residues in archaeal FttA homologues are shown in colored spheres. c, The MBL-fold of FttA is nearly structural identical to the MBL-fold of the human CPSF73 protein (PDB: 2I7T)16.

Source data

Extended Data Fig. 3 The RNA cleavage activity of FttA is stimulated by interactions with the archaeal TEC and FttA-mediated termination prefers C-rich transcripts.

Promoter-directed transcription of biotinylated templates encoding a C-less cassette permits formation of TECs with increasing length A-, G-, and U-rich nascent transcripts, respectively. FL = full-length; all templates permit elongation for 100 nts beyond the C-less cassette. b, TECs remain stably associated and transcripts are primarily recovered in the pellet (P) fraction in the absence (-) of FttA. When FttA is present ( + ), but nascent transcripts are devoid of CMP, minimal FttA-mediated transcript cleavage or termination occurs, and transcripts are not released to the supernatant (S). Lane M contains 32P-labeled ssDNA markers. Similar results were observed in 3 independent experiments and quantified (n = 3) in Fig. 2d. c, FttA demonstrates minimal RNase activity on an isolated + 125 nt transcript. Control reactions with RNaseA demonstrate that the purified transcript is not resistant to the activity of RNases. Similar results were observed in 2 independent experiments. d, Addition of T. kodakarensis RNAP to reactions containing purified + 125 nt transcripts does not stimulate FttA activity over 30 min. Similar results were obtained in two independent experiments.

Source data

Extended Data Fig. 4 The active center of FttA, an intact Spt4/5 complex and the stalk domain of the archaeal RNAP are necessary for efficient and kinetically-competitive FttA-mediated termination in vitro.

TECs+125 were assembled using promoter directed, biotinylated DNA templates. Intact TECs are bound to pellet fractions (P) while released transcripts are recovered from the supernatant (S). b, FttAH255A retains only minimal cleavage and termination activity alone, and inefficiently terminates stalled or slowly elongating TECs (lanes 17–24). FttAH255A-mediated termination becomes more efficient upon addition of Spt4-Spt5 but remains non-competitive with transcription elongation at high [NTP] (lanes 25–32). Note that the left part of this figure (lanes 1–16) is a duplication of the left part of Fig. 3c. c, Spt4/5 complexes stimulate FttA-mediated termination (Fig. 3), however, addition of Spt5 alone, containing (lanes 17–24) or lacking (lanes 25–32) the N-terminal NGN domain, fails to stimulate FttA-mediated termination to be competitive with transcription elongation at high [NTPs]. d, Spt4 alone, or together with the KOW domain of Spt5, is insufficient to stimulate FttA-mediated termination to be competitive with transcription elongation at high [NTPs]. e, While TECs assembled with RNAPWT support kinetically-competitive FttA-mediated termination (Fig. 3), TECs generated with RNAP ΔF/ΔE only support FttA-mediated termination of stalled or slowly elongating complexes. The absence of the stalk domain impairs both FttA-mediated cleavage and release of the nascent transcript, and while FttA-activity can be stimulated by the addition of Spt4/5, the hinderance to FttA-mediated termination in the absence of the stalk domain impairs FttA-mediated termination under condition of high [NTP]. Each experiment (b–e) was performed once independently.

Extended Data Fig. 5 FttA is an abundant protein likely responsible for 3′-end formation in archaeal cells.

Quantitative Western blots employing anti-FttA antibodies, purified recombinant FttA, and total cellular lysates derived from known numbers of lysed T. kodakarensis cells reveal that FttA is present at ~2,100 -/ + 500 copies per cell. Cell counts and protein calculations were performed as described20. A, B and C represent independent biological samples.

Source data

Extended Data Fig. 6 Gentle-purification of FttA directly from lysates of T. kodakarensis strain TK1428D.

Top panels show SDS-PAGE gels of fractions recovered from imidazole elutions of total cell lysates from strains TK1428D (left) and TS559 (right) resolved over 5 ml Ni2+-charged chelating columns (GE Healthcare). Bottom panels are Western blots of the same fractions from above probed with anti-HA antibodies to identify fractions within TK1428D lysates that contain FttA. The fractions pooled and analyzed by MuDPIT are identified. Magic Mark protein ladders are identified by molecular weight in Kda to the left of the gels. Data shown are from a single experiment.

Extended Data Fig. 7

Proteins identified as co-eluting partners of FttA from lysates of strain TK1428D.

Extended Data Fig. 8 FttA-mediated transcription termination completes the archaeal transcription cycle.

Promoter-directed assembly of pre-initiation complexes requires RNAP, TFB and TBP and is often assisted by TFE. De novo RNA synthesis permits promoter escape and transcription initiation factors are replaced by transcription elongation factors TFS and Spt4-Spt5. The absence of a nuclear compartment permits translation initiation and the normal coupling of the archaeal transcription and translation apparatuses throughout transcription of the gene or operon, but this coupling is disrupted by translation termination. The exposed nascent transcript likely permits loading of FttA to TECs and FttA activity mediates cleavage of nascent transcripts and release of RNAP to solution.

Supplementary Information

Supplementary Information

Supplementary Figs. 1 and 2.

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Source data

Source Data Fig. 1

The uncropped western blot used in Fig. 1e.

Source Data Fig. 4

The uncropped western blot used in Fig. 4h.

Source Data Extended Data Fig. 2

The uncropped SDS–PAGE used in Extended Data Fig. 2a.

Source Data Extended Data Fig. 3

The uncropped denaturing urea–PAGE used in Extended Data Fig. 3d.

Source Data Extended Data Fig. 5

The uncropped western blot used in Extended Data Fig. 5.

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Sanders, T.J., Wenck, B.R., Selan, J.N. et al. FttA is a CPSF73 homologue that terminates transcription in Archaea. Nat Microbiol 5, 545–553 (2020). https://doi.org/10.1038/s41564-020-0667-3

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