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Card Uses a Minor Groove Wedge Mechanism to Stabilize the RNA 1 CarD uses a minor groove wedge mechanism to stabilize the RNA 2 polymerase open promoter complex 3 4 Brian Bae1, James Chen1, Elizabeth Davis1, Katherine Leon1, Seth A. Darst1,*, 5 Elizabeth A. Campbell1,* 6 7 1The Rockefeller University, Laboratory for Molecular Biophysics, 1230 York Avenue, New York, 8 NY 10065, USA. 9 10 *Correspondence to: E-mail: [email protected], [email protected] 11 12 Present Address: Elizabeth Davis, The University of Minnesota School of Medicine, 13 420 Delaware St. SE, Minneapolis, MN 55455, USA; Katherine Leon, Department of 14 Biochemistry and Molecular Biology, University of Chicago, 929 East 57th Street, GCIS 15 W219 Chicago, IL 60637, USA. 16 17 2 18 Abstract A key point to regulate gene expression is at transcription initiation, and 19 activators play a major role. CarD, an essential activator in Mycobacterium tuberculosis, 20 is found in many bacteria, including Thermus species, but absent in Escherichia coli. To 21 delineate the molecular mechanism of CarD, we determined crystal structures of 22 Thermus transcription initiation complexes containing CarD. The structures show CarD 23 interacts with the unique DNA topology presented by the upstream double- 24 stranded/single-stranded DNA junction of the transcription bubble. We confirm that our 25 structures correspond to functional activation complexes, and extend our understanding 26 of the role of a conserved CarD Trp residue that serves as a minor groove wedge, 27 preventing collapse of the transcription bubble to stabilize the transcription initiation 28 complex. Unlike E. coli RNAP, many bacterial RNAPs form unstable promoter 29 complexes, explaining the need for CarD. 30 31 Introduction 32 Decades of research using Escherichia coli (Eco) as a model system inform most of our 33 understanding of how bacteria control transcription initiation. First, dissociable promoter 34 specificity subunits, σ factors, direct the catalytic core of the RNA polymerase (RNAP) 35 to promoter DNA sites and play a key role in unwinding the DNA duplex to create the 36 transcription bubble in the RNAP holoenzyme open promoter complex (RPo)(Feklistov 37 et al, 2014). Second, DNA-binding transcription factors either activate or repress the 38 initiation rate (Browning & Busby, 2004). 3 39 The majority of transcription activators characterized to date are dimeric proteins 40 that bind operators upstream of the promoter -35 element and directly contact the 41 RNAP α subunit (Ebright, 1993), the σ4 domain positioned at the -35 element, or both 42 (Dove et al, 2003; Nickels et al, 2002; Jain et al, 2004). Activators can accelerate 43 initiation by stabilizing the initial RNAP/promoter complex, by stimulating the 44 isomerization of the initial RNAP/promoter complex to RPo (i.e. unwinding the duplex 45 DNA to form the transcription bubble), or both (Li et al, 1997; Roy et al, 1998). 46 CarD, first identified as a regulator of ribosomal RNA (rRNA) transcription in 47 Mycobacterium tuberculosis (Mtb), is a transcriptional activator widely distributed among 48 bacterial species, including Thermus species (Stallings et al, 2009; Srivastava et al, 49 2013), but is absent in Eco (Table 1). CarD is a global regulator (Srivastava et al, 2013) 50 that is an essential protein in Mtb (Stallings et al, 2009), the causative agent of 51 tuberculosis. A deeper understanding of the CarD functional mechanism and its role in 52 the Mtb transcription program is therefore warranted. 53 Crystal structures of Tth (Srivastava et al, 2013) and Mtb (Gulten & Sacchettini, 54 2013) CarD reveal an N-terminal domain with a Tudor-like fold (CarD-RID, RNAP 55 interacting domain) in common with the Eco transcription repair coupling factor (TRCF)- 56 RID (Deaconescu et al, 2006; Stallings et al, 2009; Weiss et al, 2012), and a helical C- 57 terminal domain (CarD-CTD). Unique among known transcription activators, the CarD- 58 RID interacts with the RNAP β subunit β1-lobe (Stallings et al, 2009; Weiss et al, 2012) 59 (corresponding to the eukaryotic RNAP II Rpb2 protrusion domain; Cramer et al, 2001), 60 which is near the upstream portion of the transcription bubble in RPo (Bae et al., 2015). 61 The disposition of the CarD-CTD with respect to the CarD-RID is widely divergent in Tth 4 62 and Mtb CarD crystal structures, leading to conflicting models for the CarD activation 63 mechanism (Srivastava et al, 2013; Gulten & Sacchettini, 2013). To resolve these 64 ambiguities, we determined crystal structures of T. aquaticus (Taq) transcription 65 initiation complexes (RPo)(Bae et al., 2015) containing CarD (Figures 1A, 1B). The 66 structures show that CarD interacts with the unique DNA topology of the upstream 67 double-stranded/single-stranded (ds/ss) DNA junction of the transcription bubble. 68 Additional biochemical data confirm that our structures correspond to functional 69 activation complexes, and extend our understanding of the role of a universally 70 conserved CarD Trp residue in stabilizing the unwound transcription bubble, thereby 71 stabilizing the transcription initiation complex. 72 Throughout this work, we use three promoter sequences, full con (Gaal et al, 73 2001), Tth 23S (Hartmann et al, 1987), and Mtb AP3 (Gonzalez-y-Merchand et al, 74 1996)(Figure 1 – figure supplement 1). The full con promoter sequence, derived by 75 in vitro evolution, is likely to be optimized for binding to EσA. We use this sequence only 76 for structural studies where high-affinity, homogeneous complexes are critical for 77 crystallization. AP3 is a native Mtb rRNA promoter and its regulation by Mtb CarD has 78 been well characterized (Srivastava et al, 2013; Davis et al, 2015). In order to 79 biochemically characterize more than one promoter, we also studied 23S, a native 80 Tth rRNA promoter. In promoter-based assays, the effects of Tth or Mtb CarD on each 81 promoter were qualitatively the same. In general, we present the results from Tth 23S 82 since most of the studies used Thermus EσA and CarD. In some cases, it was 83 advantageous to use Mtb AP3 instead and we note the rationales below. 84 5 85 Results 86 Overall structure of the Thermus CarD/RPo complex 87 Crystals of CarD transcription activation complexes were prepared by soaking Tth CarD 88 into Taq Δ1.1σA-holoenzyme/us-fork(-12bp) or full RPo crystals (Bae et al., 2015). 89 Analysis of the diffraction data indicated high occupancy of one CarD molecule bound to 90 each of two RNAP/promoter complexes in the asymmetric unit of the crystal lattice 91 (Figure 1 – figure supplement 2). Docking CarD onto the RNAP was facilitated by a 92 high-resolution crystal structure of a Tth CarD/Taq β1-lobe complex (2.4 Å-resolution, 93 Table 2, Figure 1 – figure supplement 3, Figure 1 – figure supplement 4). The structures 94 of CarD transcription activation complexes were refined to 4.4 and 4.3 Å-resolution, 95 respectively (Table 1, Figure 1 – figure supplement 5). The protein/protein and 96 protein/nucleic acid interactions were essentially identical among all of the four 97 crystallographically independent complexes, so the more complete and higher 98 resolution CarD/RPo structure (Figures 1A, 1B, Figure 1 – figure supplement 5, Table 2) 99 is described here. Although the CarD bound to one RPo in the crystallographic 100 asymmetric unit made crystal-packing interactions with a symmetry-related CarD, the 101 CarD bound to the second RPo did not participate in any crystal-packing interactions 102 (Figure 1 – figure supplement 2), indicating the architecture and interactions observed 103 here are unlikely to be influenced by crystal packing interactions and likely represent the 104 functional activation complex in solution. 105 6 106 The CarD-CTD interacts with the upstream ds/ss junction of the transcription 107 bubble 108 The relative orientation of the CarD domains (CarD-RID, CarD-CTD) seen in the 109 Thermus CarD (Srivastava et al, 2013) and CarD/β1-lobe (Figure 1 – figure 110 supplement 3) structures is only slightly altered in the Thermus CarD/RPo complex: the 111 CarD-CTD is rotated ~11° (with respect to the CarD-RID) to interact with the DNA 112 (Figure 1 – figure supplement 4). By maintaining the CarD-RID/CTD interface seen in all 113 the Tth CarD structures, binding of the CarD-RID to the RNAP β1-lobe (Figures 1B, 1C, 114 Figure 1 – figure supplement 4) (Stallings et al, 2009; Weiss et al, 2012; Gulten & 115 Sacchettini, 2013) positions the CarD-CTD to interact directly with the upstream ds/ss 116 junction of the transcription bubble (Figures 1A-C). A A 117 In the RPo structure, the σ 2 and σ 3 domains make extensive interactions with 118 the promoter DNA (-17 to -4) from the (distorted) major groove side of the DNA, 119 including critical interactions that maintain the upstream ds(-12)/ss(-11) junction of the 120 transcription bubble (Figures 1A-C) (Bae et al., 2015). CarD does not make significant 121 interactions with σA but interacts with the promoter DNA from -14 to -10 from the 122 opposite, (distorted) minor groove side of the DNA (Figures 1A-C) such that the σA/DNA 123 interactions and the structure of the transcription bubble in RPo and CarD/RPo are 124 essentially the same (Figure 1 – figure supplement 6). The KMnO4 reactivity of thymine 125 (T) bases within the transcription bubble (Sasse-Dwight & Gralla, 1991; Ross & Gourse, 126 2009) is identical in the presence or absence of CarD (Figure 1D, lanes 2 and 3), 127 supporting the structural observation that the transcription bubble is the same with or 128 without CarD. Although CarD does not alter the structure of the transcription bubble, it 7 129 does increase the lifetime of RPo, as measured by the rate of disappearance of the 130 KMnO4 footprint after challenge with an excess of unlabeled competitor promoter 131 (Figure 1D, lanes 4-7) (Davis et al, 2015).
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