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Key Concepts and Challenges in Archaeal Transcription Review Key Concepts and Challenges in Archaeal Transcription Fabian Blombach, Dorota Matelska, Thomas Fouqueau, Gwenny Cackett and Finn Werner Institute of Structural and Molecular Biology, Division of Biosciences, University College London, London, WC1E 6BT, United Kingdom Correspondence to Fabian BlombachFinn Werner: [email protected], [email protected] https://doi.org/10.1016/j.jmb.2019.06.020 Abstract Transcription is enabled by RNA polymerase and general factors that allow its progress through the transcription cycle by facilitating initiation, elongation and termination. The transitions between specific stages of the transcription cycle provide opportunities for the global and gene-specific regulation of gene expression. The exact mechanisms and the extent to which the different steps of transcription are exploited for regulation vary between the domains of life, individual species and transcription units. However, a surprising degree of conservation is apparent. Similar key steps in the transcription cycle can be targeted by homologous or unrelated factors providing insights into the mechanisms of RNAP and the evolution of the transcription machinery. Archaea are bona fide prokaryotes but employ a eukaryote-like transcription system to express the information of bacteria-like genomes. Thus, archaea provide the means not only to study transcription mechanisms of interesting model systems but also to test key concepts of regulation in this arena. In this review, we discuss key principles of archaeal transcription, new questions that still await experimental investigation, and how novel integrative approaches hold great promise to fill this gap in our knowledge. © 2019 Elsevier Ltd. All rights reserved. Introduction factor NusG [5e7]. Many archaea use histones to form a chromatinized template for transcription [8,9]. Advanced phylogenetic analyses suggest that life Archaeal cells are prokaryotic, and thus, the two key on earth is divided into two primary domains, processes of gene expression, transcription and bacteria and archaea, and that a primordial translation, are likely to be functionally coupled archaeon closely related to the extant Asgård [10e12]. The transcription unit (TU) organization phylum was the likely ancestor of all eukaryotes and mRNAs of archaea and bacteria share important [1,2]. In this vein, the molecular machinery carrying basic features: (i) many genes are organized in out processing of biological information (i.e., replica- polycistronic operons, (ii) mRNAs are cap-less and tion, transcription and translation) in eukaryotes is intron-less, and (iii) translation initiation is reliant on a closely related to the cognate systems in archaea ShineeDalgarno-dependent ribosome-binding [3]. All archaeal and eukaryotic RNA polymerases mechanism or a leaderless mechanism with start (RNAPs) share a conserved core architecture in codons located proximal to the mRNA 50-terminus terms of subunit composition, structure and mole- [13]. Archaeal genomes are relatively small (1.5e7 cular mechanisms of nucleotide translocation, phos- Mbp) and compact with very short intergenic regions, phodiester bond formation and cleavage [4].In which limits the space for regulatory transcription addition, they share a core set of basal transcription factor binding sites compared to eukaryotic gen- initiation (i.e., TBP, TFB and TFE) and elongation omes. In essence, archaea run a gene expression factors (including TFS, Spt4/5, Elf1) distinct from program using a eukaryote-like transcription machin- those used by bacteria with the sole exception of ery on a bacterial-like genome. This unorthodox Spt5 that is homologous to bacterial elongation situation provides a unique opportunity to investigate 0022-2836/© 2019 Elsevier Ltd. All rights reserved. Journal of Molecular Biology (2019) 431, 4184–4201 Challenges and concepts in Archaeal transcription 4185 Fig. 1. Molecular mechanisms of the archaeal transcription cycle. (A) Transcription of a gene is initiated at the promoter, proceeds through the gene body and comes to completion at the terminator. RNAP makes repeatedly use of the template by progressing repeatedly through the cycle, and specific subsets of initiation, elongation and possibly termination factors assist RNAP during this process. These mechanisms are described in detail in the text. Atomic-resolution structural information on the RNAP transcription pre-initiation complexes (PIC) and RNAP transcription elongation complexes (TEC) has greatly contributed to our understanding of RNAP function. The structural model of the archaeal PIC (DNA-TBP-TFB- RNAP-TFEalpha) is derived from Ref. [13]. The structural model of the archaeal TEC (DNA/RNA-RNAP-Spt4/5-Elf1) was generated using Chimera. The Sulfolobus shibatae RNAP (PDB: 2WAQ) was aligned to RNAP II of the eukaryotic TEC (PDB: 5XOG, 5XON) [54,170,171]. (i) the molecular mechanisms of transcription in a The most common and well-studied archaeal model highly tractable model system (see below), (ii) the organisms for transcription exhibit an extremophilic evolution of transcription systems in archaea and lifestyle, including thermophiles such as Sulfolobus, eukaryotes, and (iii) the fundamental principles Thermococcus, Pyrococcus, and Methanocaldococ- guiding the regulation of gene expression in prokar- cus as well as the highly genetically tractable yotes and by inference eukaryotes. halophiles including Halobacterium and Haloferax 4186 Challenges and concepts in Archaeal transcription species. The thermophilic lifestyle of these archaea by the recruitment of RNAP and transcription factor often translates into a high biochemical tractability of E (TFE; TFIIE in eukaryotes) (Fig. 1AB). TBP and their proteins, which has made them favored targets for TFB recognize two promoter elements in a structure determination by crystallography [14e17] sequence-specific fashion, the TATA box and the and has facilitated the in vitro reconstitution of RNAPs B-recognition element (BRE), respectively. The from their 12 individual recombinant subunits [18,19],a binding of TBP distorts the promoter topology by feat that has not been successful for any eukaryotic inducing a ~90 bend in the DNA [14,36]. TFB RNAP. Reconstituted recombinant RNAPs have enhances the stability of the TBPeDNA complex allowed a rigorous molecular genetics analysis of [36], provides the correct orientation [37] and recruits archaeal RNAPs unconstrained by cell viability, as well the RNAP via multiple interactions with the RNAP as the site-specific incorporation of molecular probes dock domain and RNAP active site cleft [38,39]. TFE including fluorescent dyes and nitroxide spin labels facilitates the conversion of the PIC from the closed into RNAP subunits. These derivatized RNAPs have complex (CC) to the open complex (OC). During the been applied in a broad range of functional and conversion to the OC, the DNA strands are locally biophysical studies that have furthered our under- separated upstream of the transcription start site standing of RNAP mechanisms [19e25]. The extreme (TSS) and the template strand is inserted into the physical and chemical conditions including high active site of RNAP. The Initiator (Inr) promoter temperature and low pH require special adaptations element is a dinucleotide motif that is important for of biochemical assays and often rule out the use of the precise choice of TSS [40]. Transcription starts conventional selective agents including antibiotics and with the synthesis of very short (3e9 nt) “abortive” chemical drugs, which rapidly degrade in the growth transcripts, during which the PIC converts into the medium or reaction buffers. In addition, the archaeal initially transcribing complex (ITC) that remains RNAP appears insensitive to alpha-amanitin and bound to the promoter via TBP and TFB. During rifampicin that are widely used as inhibitors of initial transcription archaeal RNAP remains bound to eukaryotic RNAPII and bacterial RNAP, respectively the initiation factors upstream while downstream [26,27]. Many archaeal proteins are modified by DNA is railed in. This likely results in DNA scrunch- phosphorylation and methylation, but to date, there is ing as has been observed for bacterial RNAP [41].In no direct evidence that components of the archaeal order to productively synthesize RNA transcripts, the transcription machinery, including histones, are regu- RNAP has to be released from TBP and TFB. The lated by post-translational modifications [28e32].In underlying molecular mechanisms are complex and comparison, post-translational modifications play a likely involve a concerted displacement of TFB major role in the regulation of RNAPII, including the domains by conformational changes in the RNAP extensive phosphorylation of the largest RNAPII and by the growing RNA chain similar to eukaryotic subunit C-terminal domain that is not conserved in RNAPII [20,38,42]. During initiation, DNA melting archaea [28,33]. and template strand loading can occur in the Archaeal model systems have provided many absence of TFE, since TBP and TFB are sufficient insights into the fundamental mechanisms of transcrip- to initiate RNAP transcription in a TSS-specific tion. The key strategy to unlock the remaining mysteries fashion in vitro [18,43]. To what extent transcription is to integrate (i) mechanistic hypotheses derived from initiation depends on TFE in vivo remains to be molecular structures and in vitro transcription experi- determined. The essential nature of tfeA, the gene ments, (ii) classical
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