Transcription Regulation in Archaea

43 Pages·2016·0.85 MB·English

JB Accepted Manuscript Posted Online 2 May 2016
J. Bacteriol. doi:10.1128/JB.00255-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 Title: Transcription Regulation in Archaea
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3 Authors: Alexandra M. Gehring, Julie E. Walker, Thomas J. Santangelo#
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5 Affiliation: Department of Biochemistry and Molecular Biology
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6 Colorado State University w
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7 Fort Collins, CO, 80523, USA a
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9 Running Title: Transcription Regulation in Archaea o
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10 Corresponding Author: Thomas J. Santangelo t
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11 1870 Campus Delivery, 383 MRB //
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12 Colorado State University a
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13 Fort Collins, CO, 80523, USA .o
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14 Tel: [phone] /
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15 [email protected] D
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17 Footnote: A.M.G. and J.E.W. contributed equally to this work. e
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19 Abstract
20 The known diversity of metabolic strategies and physiological adaptations of archaeal species to
21 extreme environments is extraordinary. Accurate and responsive mechanisms to ensure that
22 gene expression patterns match the needs of the cell necessitate regulatory strategies that
23 control the activities and output of the archaeal transcription apparatus. Archaea are reliant on
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24 a single RNA polymerase for all transcription and many of the known regulatory mechanisms w
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25 employed for archaeal transcription mimic strategies also employed for eukaryotic and bacterial a
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26 species. Novel mechanisms of transcription regulation have become apparent by increasingly d

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27 sophisticated in vivo and in vitro investigations of archaeal species. This review emphasizes o
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28 recent progress in understanding archaeal transcription regulatory mechanisms and highlights t
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29 insights gained from studies of the influence of archaeal chromatin on transcription. //
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31 Introduction
32 RNA polymerase (RNAP) is a well-conserved, multi-subunit essential enzyme that
33 transcribes DNA to generate RNA in all cells. Although RNA synthesis is carried out by RNAP,
34 the activities of RNAP during each phase of transcription are subject to basal and regulatory
35 transcription factors. Substantial differences in transcription regulatory strategies exist in the
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36 three Domains (Bacteria, Archaea, and Eukarya). Only a single transcription factor (NusG or w
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37 Spt5) is universally conserved (1, 2), and the roles of many archaeal-encoded factors have not a
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38 been evaluated using in vivo and in vitro techniques. Archaea are reliant on a transcription d

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39 apparatus that is homologous to the eukaryotic transcription machinery; similarities include o
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40 additional RNAP subunits that form a discrete subdomain of RNAP (3, 4), as well as basal t
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41 transcription factors that direct transcription initiation and elongation (5–8). The shared //
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42 homology of archaeal-eukaryotic transcription components aligns with the shared ancestry of a
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43 Archaea and Eukarya, and this homology often is exclusive of Bacteria. Archaea are .o
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44 prokaryotic but the transcription apparatus of Bacteria differs significantly from that of Archaea /
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45 and Eukarya. D
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46 The archaeal transcription apparatus is most commonly summarized as a simplified e
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47 version of the eukaryotic machinery. In some respects this is true, as homologs of only a few e
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48 eukaryotic transcription factors are encoded in archaeal genomes and archaeal transcription in 1
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49 vitro can be supported by just a handful of transcription factors. However, much regulatory 0
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50 activity in eukaryotes is devoted to post-translational modifications of chromatin, RNAP, and b
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51 transcription factors, and this complexity seemingly does not transfer to the archaea where few g
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52 post-translational modifications or chromatin-imposed regulation events are currently known. t
53 The ostensible simplicity of archaeal transcription is under constant revision as more detailed
54 examinations of archaeal-encoded factors become possible through increasingly sophisticated
55 in vivo and in vitro techniques. This review will highlight the current understanding of archaeal
56 transcription, emphasizing the roles of factors that regulate archaeal RNAP throughout each
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57 stage of the transcription cycle and also highlighting outstanding issues in the field.
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59 The archaeal transcription cycle
60 Transcription is highly regulated and the transcription cycle is typically demarcated into
61 three phases: initiation, elongation and termination (9–13) (Figure 1). An abbreviated and
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62 overall introduction to this cycle is presented first, with sections below detailing the activities of w
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63 RNAP and associated factors during each stage of transcription. Briefly, archaeal transcription a
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64 initiation requires that RNAP be directed to promoter-sequences defined by the binding of TATA d

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65 binding protein (TBP) and transcription-factor B (TFB). TBP, TFB, and RNAP are sufficient to o
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66 generate a single-stranded section of DNA (the transcription bubble) and feed the template t
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67 strand into the bi-partite active center of RNAP (7, 14). RNAP can initiate transcript synthesis //
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68 de novo and continued synthesis then competes with favorable promoter and initiation factor a
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69 contacts until promoter escape can be achieved. Release of RNAP from the initiating factors .o
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70 classically defines the end of initiation, although in reality no clear boundary separates the latter /
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71 stages of initiation from the early stages of elongation. Although TFB and TBP are necessary D
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72 and sufficient to permit promoter-directed transcription initiation, a third conserved factor, e
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73 transcription factor E (TFE), can also assist in transcription initiation and likely leaves the e
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74 promoter with RNAP during the early stages of transcript elongation (15–18). Transition to a 1
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75 stable, long-lived elongation complex is believed to involve internal rearrangements of RNAP. 0
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76 This transition involves the exchange of initiation factors for stably bound elongation factors that b
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77 monitor RNA synthesis for accuracy, respond to regulatory DNA sequences, react to regulatory g
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78 inputs of more transiently associated transcription factors, and influence processivity of RNAP. t
79 Elongation is, in general, very stable, but specific sequences can lower the overall energy of the
80 transcription elongation complex permitting either spontaneous intrinsic or factor-assisted
81 termination (19, 20). Transcription termination results in release of both the transcript and
82 RNAP from the DNA template.
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84 Regulated transcription initiation
85 Transcription initiation is tightly regulated by both transcription factors and DNA
86 elements. The minimal, necessary proteins and DNA elements for archaeal transcription
87 initiation are now well defined and characterized (21–28). A recent excellent review (29) D
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88 summarizes the actions of repressors and activators that function during initiation in archaeal w
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89 species. We focus here on the roles of new DNA elements and newly discovered strategies of a
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90 basal initiation factors. d

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91 Basal Transcription Factors m

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92 TBP and TFB are the only transcription factors required for in vitro transcription under t
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93 optimized conditions, and TFE has been shown to assist promoter-opening when conditions are /jb
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94 sub-optimal (16). In vivo studies have shown that archaea must retain at least one gene s
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95 encoding TBP and one gene encoding TFB, although many archaeal species encode multiple o
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96 TBP and TFB isoforms (6, 21, 30–35). Some differences in promoter-sequence preferences and o
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97 protein-pairing have been noted in TBP-TFB isoform pairs (36–41), but these minor differences D
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98 are not on par with the clear but not always radical promoter-sequence differences noted for e
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99 alternative σ-factors in bacterial transcription (39, 42). TFE also appears essential, and it is e
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100 currently unclear if this essentiality is due to necessary activities during transcription initiation or 1
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101 some other role in the transcription cycle (26, 43, 44). 0
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102 All three of the aforementioned transcription factors have close eukaryotic homologs: b
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103 archaeal TBPs are nearly identical to eukaryotic TBPs (45); archaeal TFB proteins are u
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104 homologous to eukaryotic TFIIB proteins (46), with homology also seen with the Pol III initiation t
105 factor BRF1 (47) and Pol I initiation factor Rrn7/TAF1B (48); and archaeal TFE proteins are
106 homologous to the N-terminal half of the eukaryotic TFIIEα, and very recent evidence identified
107 a separate homolog in some lineages to eukaryotic TFIIEβ (17). TBP is needed to recognize the
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108 TATA box, bend the DNA, and recruit TFB (46); its role had therefore been deemed equivalent
109 to the role of eukaryotic TBPs. Recent, sophisticated TIRF-FRET measurements now detail
110 differences in the activities of archaeal and eukaryotic TBPs, despite the nearly identical 3-D
111 folds of these factors (6). In some cases, archaeal TBPs require the co-binding of TFB to stably
112 bind and bend the promoter DNA (6, 22, 49, 50). It is tempting to speculate that different
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113 promoter sequences may be regulated by different TFB-TBP pairs based on the w
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114 interdependence, or lack thereof of cooperative DNA bending for establishing a stable platform a
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115 for RNAP recruitment. Recent studies suggest that select isoforms of TFB and TBP can result d

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116 in differences in transcription output, but further studies will be needed to determine if these o
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117 effects on such preliminary steps of transcription initiation are a direct mode of regulation t
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118 resulting in phenotypic differences (37, 51). //
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119 In contrast to eukaryotic transcription, archaeal promoter opening is not an energy- a
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120 dependent process (7). Therefore, TBP and TFB alone are capable of assisting RNAP in .o
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121 formation of the transcription bubble. In all archaea, TFB is responsible for stabilizing the TBP /
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122 bound DNA complex and together this bi-partite protein platform recruits RNAP (52), but how D
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123 these molecular interactions melt the DNA is still unresolved. Reconstructions and analyses of e
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124 open complexes using archaeal components reveal an overall architecture of the open promoter e
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125 complex and provides the first placement of the non-template strand within the complex (52). 1
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126 TBP and TFB are located closer to RNAP than would be the case for eukaryotic promoters and 0
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127 this proximity may provide more intimate contacts that collectively provide the energy to open b
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128 the promoter DNA. The tight network of interactions in the archaeal open complex may g
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129 torsionally strain the DNA and melting is likely to reveal this strain and result in open complex t
130 formation.
131 Several new insights into TFE activity and evolution have been recently described.
132 Archaeal TFE had previously been characterized as a monomer and as a homologue of the
133 alpha-subunit of eukaryotic TFIIE, termed TFIIEα (16, 18, 53). Eukaryotic TFIIE is a
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134 heterodimeric complex of TFIIEα and TFIIEβ, but archaeal genomes had previously only been
135 shown to encode a homologue of the alpha-subunit (54, 55). Eukaryotic RNAPs differ in their
136 requirements for initiation, with RNAP III incorporating homologues of several RNAP II initiation
137 factors as core components of RNAP III (56–58). Comparisons of the RNAP III subunit hRCP39
138 revealed a well-conserved archaeal homolog (termed TFEβ) that directly and extensively
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139 interacts with TFE (now named TFEα) (17). Although TFEβ is not conserved in all archaea, w
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140 TFEβ is essential for some crenarchaea; when employed in vitro, TFEα/TFEβ complexes are a
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141 effective in binding RNAP, stabilizing open complex formation, and stimulating total d

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142 transcriptional output (17). o
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143 The mechanism of TFE recruitment to the initiation complex and its activities during t
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144 initiation have been partially resolved. TFEα simultaneously binds TBP, RNAP and downstream //
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145 DNA, and has been shown to stimulate transcription at non-canonical promoter sequences and a
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146 at reduced temperatures in vitro (16, 18, 59). Several studies have identified critical interactions .o
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147 between TFE and the pre-initiation complex that have furthered our understanding of TFE /
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148 function during initiation (2, 15, 26, 53, 59). TFEα consists of two domains: a winged helix (WH) D
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149 domain and a zinc-ribbon (ZR) domain (60, 61); TFEβ contains a conserved WH domain and a e
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150 FeS domain (17). The winged-helix domain of TFEα contacts the upstream, non-template strand e
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151 of DNA and helps form the open-promoter complex through an unknown mechanism (15, 52). 1
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152 Several studies have shown that the presence of the RNAP stalk domain, unique to archaeo- 0
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153 eukaryotic RNAPs and comprised of two subunits – RpoE and RpoF in archaea and Rpo4 and b
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154 Rpo7 in eukaryotes – is essential for the full activity of TFEα (59, 62, 63). The predicted g
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155 interaction between TFEα and the stalk domain was bolstered by co-purification of TFEα with t
156 intact RNAP but the loss of TFEα from RNAP preparations wherein the stalk domain was
157 missing (44). A recent structure-function study identified critical interactions between TFEα and
158 RpoE of the stalk domain (26). TFE may have an essential role in modulating intramolecular
159 movements of RNAP during the transcription cycle, most notably movements of the clamp
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160 domain. Interaction of TFEα with both the stalk and clamp domains of RNAP during
161 transcription initiation may retain the clamp domain in an open conformation necessary for
162 initiation and early elongation. Replacement of TFE by Spt4/5 during early elongation may alter
163 clamp positioning and further stabilize the elongation complex (2).
164 DNA elements
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165 Transcription initiation is regulated by DNA elements that are both recognized by basal w
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166 transcription factors and that influence subsequent steps in promoter opening. There are four a
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167 DNA elements currently known to regulate archaeal transcription initiation; i) the TATA box d

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168 located approximately 25 bp upstream of the site of transcription initiation (64–66), ii) the TFB o
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169 Recognition Element (BRE) located immediately upstream of the TATA box (6), iii) the initiator t
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170 element (INR) located within the initially transcribed region, and iv) promoter proximal element //
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171 (PPE) located between the TATA box and the site of transcription initiation (67–69). Of these a
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172 four, only the TATA box and BRE are required for transcription initiation, although alterations to .o
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173 all four elements can influence total output of a promoter. /
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174 The INR is not a required DNA element for transcription initiation; however, it is a D
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175 regulatory element that can increase the strength of the promoter in a TATA- and BRE- e
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176 dependent manner. The INR is a core promoter element located in the 5′ untranslated region e
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177 (UTR), and has sequence similarity to the TATA box. The INR has been shown to be targeted 1
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178 by some transcriptional activators, and its high A/T content may facilitate promoter-opening in 0
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179 some instances. Given that many archaeal transcripts are leaderless, the INR is not b
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180 consistently identifiable, and the regulatory influence of INR sequences does not appear to g
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181 extend to RNA half-life or altering translational capacity (70). PPEs, centered at approximately t
182 ten bps upstream of the site of initiation, have been shown to increase transcription output
183 through recruitment of TFB (67, 68). Additionally, permanganate footprinting data of the
184 preinitiation complex, demonstrated that the border of the transcription bubble is at the PPE and
185 that this regions is important for the activity of TFEα/β (17).
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187 Regulation of Elongation
188 As transcription transitions from initiation to elongation, RNAP undergoes a
189 conformational change accompanied by the replacement of initiation factors with elongation
190 factors (2, 12, 71–74). It is plausible that the emerging nascent transcript stimulates the swap
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191 of regulatory factors and initiates the intramolecular movements that result in stable elongation w
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192 complex formation (75, 76). Very few transcription elongation factors have been a
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193 bioinformatically identified within archaeal genomes and it is probable that archaeal-specific d

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194 factors await discovery. It is noteworthy what is seemingly not encoded in archaeal genomes, o
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195 given that so much of archaeal and eukaryotic transcription machinery is shared. Archaeal t
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196 genomes do not appear to encode any co-activator complexes nor mega-complexes for //
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197 chromatin-modification or rearrangements. There does not appear to be machinery for a
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198 regulated post-translational modifications of the archaeal transcription apparatus nor chromatin, .o
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199 with the exception of acetylation/deacetylation of the small chromatin-associated protein Alba /
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200 (77–80). Furthermore, archaeal transcripts are not capped, do not require nuclear export and D
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201 with the exception of self-splicing introns, are intron-less; thus factors responsible for these e
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202 activities are similarly lacking from archaeal genomes (81–83). e
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203 Transcription elongation factors have various roles including increasing processivity and 1
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204 fidelity of RNAP and/or increasing genome stability. Only two archaeal elongation factors have 0
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205 been experimentally studied: the aforementioned universally conserved elongation factor Spt5, b
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206 often with a conserved binding partner Spt4 (Spt4/5) (2, 84, 85) and transcription factor S (TFS) g
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207 (86, 87). Several recent studies have shed light onto the roles of Spt5 during elongation (1, 72, t
208 88, 89). TFS, with homology to the C-terminal domain of eukaryotic TFIIS and functionally
209 analogous to GreA/GreB in Bacteria (8, 90–92) can stimulate endonucleolytic cleavage of the
210 RNA from backtracked RNAP complexes (86, 92–94). The finding of multiple TFS homologs in
211 some archaeal lineages offers the possibility of unique regulatory roles of specific isoforms.
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212 Transcription factor Spt5
213 Archaeal Spt5, homologous to bacterial-encoded NusG, consists of two domains: the
214 NusG N-terminal (NGN) domain and a single, C-terminal Kyrpides-Ouzounis Woese (KOW)
215 domain with affinity for single stranded RNA (84, 85, 88); eukaryotic Spt5 typically contain three
216 to six repeats of the C-terminal KOWb domain (95–97). Critical, direct molecular interactions
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217 between Spt5 and RNAP have been identified in both Bacteria and Archaea (84, 85, 88, 89, 96, w
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218 98–100), and the conservation of RNAP and Spt5 infers that these same interactions are used a
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219 in Eukarya. Briefly, a hydrophobic depression on the NGN domain interacts with the mobile d

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220 clamp domain of RNAP, with additional interactions between the NGN domain and RNAP jaw o
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221 domain likely fixing the location of the clamp domain in a closed configuration (11, 99). Spt5 t
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222 interaction with RNAP is not necessary for productive and processive elongation in vitro, but the //
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223 interaction does increase total output of transcription systems (1). It is plausible that Spt5 a
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224 increases elongation rates and processivity, as E. coli NusG does, and it is further possible that .o
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225 the increased efficiency of transcription results from the stabilization of the clamp domain that in /
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226 turn stabilizes the DNA:RNA hybrid in place during transcription elongation (88, 101–103). The D
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227 NGN domain also contacts the upstream strands of DNA offering protection from backtracking e
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228 and, by inference, may reduce pausing of the transcription elongation complex (88, 89, 104, e
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229 105). It is of importance to note that NusG/Spt5 can have a positive and/or negative effect on 1
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230 elongation rates and pause events of RNAP. In Thermus thermophilus NusG slows down RNA 0
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231 elongation rather than increases elongation rates (106). In Bacillus subtilis sequence-specific b
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232 interactions of the NGN and non-template DNA strand within the paused transcription bubble g
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233 stabilized the pause event in the trp operon (104, 107). Furthermore, evidence has shown that t
234 Spt4/5 induces pauses during early elongation of pol I but promotes elongation downstream
235 (108). Although NusG can elicit opposite roles on transcription elongation, the NusG:RNAP
236 binding sites remain well-conserved across various species. Archaeal and eukaryotic-genomes
237 often encode an additional elongation factor, Spt4 (annotated as RpoE’’/RpoE2 in archaea), that
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