FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3031290989> ?p ?o ?g. }

• W3031290989 abstract "Article2 June 2020free access Source DataTransparent process Structural basis of bacterial σ28-mediated transcription reveals roles of the RNA polymerase zinc-binding domain Wei Shi Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Wei Zhou orcid.org/0000-0001-9508-3598 Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Baoyue Zhang Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Shaojia Huang Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yanan Jiang Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China Search for more papers by this author Abigail Schammel Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Yangbo Hu Corresponding Author [email protected] orcid.org/0000-0001-6153-1348 Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Bin Liu Corresponding Author [email protected] orcid.org/0000-0002-6581-780X Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Wei Shi Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Wei Zhou orcid.org/0000-0001-9508-3598 Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Baoyue Zhang Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Shaojia Huang Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China University of Chinese Academy of Sciences, Beijing, China Search for more papers by this author Yanan Jiang Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China Search for more papers by this author Abigail Schammel Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Yangbo Hu Corresponding Author [email protected] orcid.org/0000-0001-6153-1348 Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China Search for more papers by this author Bin Liu Corresponding Author [email protected] orcid.org/0000-0002-6581-780X Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA Search for more papers by this author Author Information Wei Shi1,‡, Wei Zhou2,3,‡, Baoyue Zhang2,3, Shaojia Huang2,3, Yanan Jiang1,4, Abigail Schammel1, Yangbo Hu *,2 and Bin Liu *,1 1Section of Transcription & Gene Regulation, The Hormel Institute, University of Minnesota, Austin, MN, USA 2Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China 3University of Chinese Academy of Sciences, Beijing, China 4Department of Pathophysiology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 27 87199354; E-mail: [email protected] *Corresponding author. Tel: +1 507 437 9646; E-mail: [email protected] EMBO J (2020)39:e104389https://doi.org/10.15252/embj.2020104389 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In bacteria, σ28 is the flagella-specific sigma factor that targets RNA polymerase (RNAP) to control the expression of flagella-related genes involving bacterial motility and chemotaxis. However, the structural mechanism of σ28-dependent promoter recognition remains uncharacterized. Here, we report cryo-EM structures of E. coli σ28-dependent transcribing complexes on a complete flagella-specific promoter. These structures reveal how σ28-RNAP recognizes promoter DNA through strong interactions with the −10 element, but weak contacts with the −35 element, to initiate transcription. In addition, we observed a distinct architecture in which the β′ zinc-binding domain (ZBD) of RNAP stretches out from its canonical position to interact with the upstream non-template strand. Further in vitro and in vivo assays demonstrate that this interaction has the overall effect of facilitating closed-to-open isomerization of the RNAP–promoter complex by compensating for the weak interaction between σ4 and −35 element. This suggests that ZBD relocation may be a general mechanism employed by σ70 family factors to enhance transcription from promoters with weak σ4/−35 element interactions. Synopsis Cryo-EM structures of a σ28-transcription initiation complex (TIC) reveal a novel role of β′ bacterial RNA polymerase (RNAP) zinc-binding domain (ZBD) in transcription initiation. Relocation of the ZBD may compensate for weak σ4/−35 element interactions and hereby enhance transcription from this type of promoters. Cryo-EM reconstructions of σ28-TIC at 3.86 Å (state 1) and 3.91 Å (state 2) define the specific promoter recognition by σ28. Cryo-EM structure of state-2 σ28-TIC reveals a distinct architecture of β′ zinc binding domain of RNAP. ZBD relocation has an overall effect of facilitating the isomerization of RNAP-promoter closed to open complex to enhance transcription. ZBD relocation could be exploited by the σ70-family factors to enhance Transcription by compensating for the weak interaction between σ4 and promoter −35 element. Introduction Multi-subunit DNA-dependent RNA polymerase (RNAP) is the core enzyme responsible for transcription, the first step of gene expression in cells. In bacteria, the RNAP core enzyme comprises four types of evolutionarily conserved subunits (α2ββ′ω) (Murakami & Darst, 2003; Borukhov & Nudler, 2008), which is unable to recognize specific promoter sequences alone. Transcription initiation is tightly regulated by sigma factors, in which the core enzyme first binds a specific sigma factor to form a holoenzyme and then recognizes specific promoters. The subsequent step is isomerizing from closed RNAP–promoter complex (RPc) to open RNAP–promoter complex (RPo) with melted base pairs (bp) that is competent to initiate transcription (Feklistov & Darst, 2011; Saecker et al, 2011; Ruff et al, 2015; Browning & Busby, 2016). On the basis of the structural and functional differences, bacterial sigma factors could be classified into two main families: the primary σ70 factor family and the σ54 factor family for nitrogen regulation and some stress responses (Feklistov et al, 2014). The σ70 family factors are then subdivided into four major groups according to the different compositions of the conserved domains—σ1.1 (σR1.1 region), σ2 (σR1.2, σNCR, and σR2.1–2.4 regions), σ3 (σR3.0 and σR3.1 regions), and σ4 (σR4.1 and σR4.2 regions) (Feklistov et al, 2014; Paget, 2015). Sigma factors in group 1 (σ70 in Escherichia coli) contain all the conserved domains, while group 2 sigma factors (σS or σ38 in E. coli) lack σNCR region, group 3 sigma factors (σ28 in E. coli, also known as RpoF or FliA) lack σR1.1, σNCR, and σR1.2 regions, and group 4 (extracytoplasmic function, ECF) sigma factors are the most stripped-down version, possessing only two essential domains σ2 and σ4 (Osterberg et al, 2011; Paget, 2015). As a representative group 3 sigma factor, σ28 is the most widely distributed alternative sigma factor that controls flagellum biosynthesis in all motile Gram-negative and Gram-positive bacteria (Paget, 2015), and is indispensable for motile bacteria to compete with other microorganisms and survive in the adverse conditions like poor nutrition (Zhao et al, 2007). In addition, σ28 has been reported to play a role in cell development in some non-motile bacteria (Chater et al, 1989; Yu & Tan, 2003). Interestingly, σ28 homologues from distant species may work identically and can successfully restore the motility in E. coli fliA mutant (Chen & Helmann, 1992; Heuner et al, 1997; Studholme & Buck, 2000). Bioinformatic analysis of σ28 promoters suggests that consensus sequences for −35 and −10 elements are TAAAGTTT and GCCGATAA, respectively, which are separated by an 11-bp spacer (Yu et al, 2006b). This distinguishing promoter feature was confirmed by mutational and biochemical analyses (Yu et al, 2006a; Koo et al, 2009; Hollands et al, 2010), and the first two nucleotides in −10 element were further defined as an extended −10 motif (Koo et al, 2009). Comparing with regulation to σ70-dependent promoters, cyclic AMP receptor protein binds to σ28-dependent promoters at an atypical location, suggesting that recognition of σ28 to −35 element may be different from σ70 (Hollands et al, 2010). Nevertheless, the molecular mechanism of σ28-dependent transcription initiation remains largely uncharacterized. The well-studied transcription initiation complex (TIC) structures of group 1 sigma factors (Murakami et al, 2002b; Zhang et al, 2012; Zuo & Steitz, 2015) and the recent TIC structures of group 2 sigma factor (Liu et al, 2016) and group 4 sigma factors (Li et al, 2019; Lin et al, 2019) have greatly facilitated our understanding of how these sigma factors recognize respective promoter elements and initiate transcription. However, as yet, there is no structure of RNAP holoenzyme or transcription complex for group 3 sigma factor—σ28—to illustrate the mechanism of transcription initiation. In this study, we assembled the intact functional E. coli transcribing complex with the flagella-specific sigma factor—σ28—and determined the first TIC structures of group 3 factors at around 3.9 Å resolution. The structures show that σ28-RNAP has strong interaction with promoter −10 element but weak contact with −35 element. Intriguingly, the β′ zinc-binding domain (ZBD), also previously known as zinc ribbon region (ZNR) (Lane & Darst, 2010), of RNAP stretches out from its canonical position to interact with non-template strand (NT-strand) in a distinct architecture, which consequently stabilizes promoter binding to advance transcription initiation by compensating for the weak interaction between σ4 and −35 element. Further in vitro and in vivo tests reveal that ZBD relocation shows an overall effect of facilitating the isomerization from RNAP–promoter closed complex to open complex, and also suggest that the relocation of β′ ZBD is a general mechanism employed by sigma factors with weak σ4/−35 element interactions in transcription initiation. These observations advance our understanding of transcription initiation by RNAP from weakly bound promoters, such as promoters with non-conserved −35 element, which comprise the majority in bacteria (Ettwiller et al, 2016). Results Overall structure of the Escherichia coli σ28-dependent transcribing complex To obtain the cryo-EM structure of the E. coli σ28-dependent transcribing complex, we assembled the complex with RNAP core enzyme, σ28 factor, the synthetic DNA scaffold (from −39 to +15, positions relative to the transcription start site +1) (Fig 1A). Incubation with nucleotides (ATP and CTP) and Mg2+ produced transcription initiation complexes (TICs) with a nascent AAC RNA (Fig EV1A and B). Figure 1. Cryo-EM reconstructions of the σ28-TICs at the two states Schematic representation of the synthetic promoter DNA scaffold (54-bp) in the σ28-TICs. The −10 and the extended −10 elements were annotated by a dashed box according to the previous report (Koo et al, 2009) and shown in color letters based on the observed structure in this study. The predicted −35 element (Yu et al, 2006b) and the observed one were also annotated in the same way. Overviews of the cryo-EM reconstruction maps of the E. coli σ28-TICs at the state 1 (left, 3.86 Å resolution) and state 2 (right, 3.91 Å resolution), respectively. The individually colored density maps, created by color zone and split in Chimera and contoured at 1.0 of view value of Chimera, are displayed in transparent surface representation to allow visualization of all the components of the complex. Zoom-in views of β′ ZBDs in the σ28-TICs. The split density map for the β subunit was omitted for clear representation. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Isolation of the σ28-TIC, cryo-EM images, and data processing procedure for σ28-TIC A. The size-exclusion chromatography profile of the σ28 -TIC is presented. Peak 2 is the target complex, while peaks 1, 3, and 4 are the aggregation form, surplus DNA, and surplus NTP, respectively. B. The SDS–PAGE gel visualized the components and verified the presence of the complex. C. A representative micrograph. The scale bar size is 36.6 nm. D. Flow chart of the cryo-EM image processing (see Method details). E. Selected 2D classes for the structure 0. F. Gold-standard Fourier shell correlations (FSCs) of the maps for structures 0–2. G, H. Angular orientation distributions of the particles used in the final reconstruction (left) and FSC curves of half maps and model to map (right) for structure 1 (state-1 σ28-TIC) (G) and structure 2 (state-2 σ28-TIC) (H). Source data are available online for this figure. Download figure Download PowerPoint The cryo-EM single-particle reconstruction of the complex showed two different architectures corresponding to two states (Fig 1B and C). The state 1 and state 2 structures were reconstructed at the overall resolutions of 3.86 Å and 3.91 Å, respectively (Fig EV1C–H, and Appendix Table S1). The qualities of map reconstruction evaluated using Mtriage in Phenix showed the resolutions of 4.23 Å (state 1) and 4.27 Å (state 2) according to half-map Fourier shell correlation (FSC) calculations at the criteria of 0.143 (Fig EV1G and H). Further local resolution maps displayed the resolution ranges for the individual parts of the complex: most parts of RNAP at 3–4.25 Å, β′ ZBD at 3–5.5 Å, and major parts of σ28 and DNA scaffold at 3–5.5 Å, in which the areas involving protein–DNA interactions have better local resolutions, including β′ ZBD and DNA-binding regions in σ28 (Fig EV2). The local real-space correlation coefficients (CC) for the ZBD domain calculated at the resolution of 5 Å using Chimera are 0.8872 (6PMI, state 1) and 0.8367 (6PMJ, state 2), respectively. The densities in the cryo-EM maps were well fitted by the core RNAP, which shows a similar overall architecture to that observed in other TICs (Zuo & Steitz, 2015; Liu et al, 2016, 2017). The σ28 factor has relatively lower sequence identity (22.7%) and similarity (38.9%) to σ70 factor (Fig EV3A). However, the folding pattern of σ28 factor in TICs shows high similarity with that of σ38 or σ70 throughout the conserved regions, from σR2.1 to σR4.2 regions, indicating the conserved structures of the σ70 family in TICs. Most of the side chains of amino acids in σ28 are visible in the density map, suggesting good modeling reliability (Fig EV3B). The structures of the two states also display a well-ordered density of nucleic acids including the bubble region and the newly synthesized RNA (Fig EV3C and D). Click here to expand this figure. Figure EV2. Local resolution maps for σ28-TIC A, B. The overall local resolution map (top) and the local resolution maps for individual parts (bottom) of structure 1 (state-1 σ28-TIC) (A) and structure 2 (state-2 σ28-TIC) (B). The local resolution map is contoured at 1.0 of view value of Chimera. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Alignments of the σ28 factor with other factors and the nucleic acid structures in σ28-TICs A. The sequence alignment of the σ28 factor with the σ38, σ70 factor in E. coli. Sequences are presented with the one-letter amino acid codes. The second structure (α helices) and the conserved regions are labeled above and below the sequences, respectively. The black triangles are used to denote the promoter-recognition amino acids of σ28 factor. B. Overall structure of the E. coli σ28 factor in the TICs. The side chains are shown as stick mode, and the transparent density map is contoured at 1.0 of view value of Chimera (left). The E. coli σ28 factor in the σ28-TIC, the σ38 factor in the σ38-TIC (PDB ID 5IPL), and the σ70 factor in the σ70-TIC (PDB ID 4YLN) are superimposed (right). C, D. The transparent split cryo-EM maps (contoured at 1.0 of view value of Chimera) for the promoter DNA, the nascent RNA, and the active site Mg ion in the σ28-TIC at the state 1 (C) and the state 2 (D) are shown, respectively. The red labeled regions on the promoter DNA are the important recognition regions. The color schemes for others are the same as in Fig 1. The right insets are the zoom-in views of the DNA-RNA hybrid. Download figure Download PowerPoint The two structures of σ28-TIC complexes show high similarity, with a root-mean-square deviation (RMSD) of 0.255 Å (Cα aligned). The major difference in two structures lies in the location of β′ ZBD of RNAP. In state 1 structure, ZBD is adjacent to the σ4 domain and does not interact with the upstream DNA; in state 2 structure, ZBD gets close to the upstream DNA and makes contact with the NT-strand DNA (Fig 1B and C). Stable promoter recognition on the −10 element but weak on the −35 element The σ28 factor recognizes the −10 and −35 elements via its σ2 and σ4 domains, respectively, which is similar to other σ70 family factors in TIC structures (Bae et al, 2015; Zuo & Steitz, 2015). The strong density on the −10 element suggests a stable recognition here. Five positively charged arginine residues of σ28 have pairs of polar interactions with nucleotides of the template −10 element: R95/−13G, R95/−12C, R98/−12C, R98/−11T, R84/−12C, R34/−11T, and R94/−11T (Fig 2A and B), which explains previous observation that single mutation in R84 or R98 significantly decreased σ28-dependent transcription (Koo et al, 2009). In addition, several residues in the β and β′ subunits (βN494, βK496, βR470, β′S319, and β′R259) also make contacts with the template strand (T-strand) −10 element via hydrogen bond or polar interactions (Fig 2A and B), which are similar to those observed in the σ38-TIC (Liu et al, 2016). The NT-strand −10 element is contacted by the region of σR2.3, and the path down to the main cleft is the same as that in previously reported TICs (Feklistov & Darst, 2011; Zhang et al, 2012). Multiple residues of the σR2 region contribute to the recognition of the sequence: R58/−13C, R74/−13C, Q73/−12G, Q63/−11A, T69/−9A, and H26/−7C (Fig 2A and B). In addition to recognizing the core −10 element, σ28 also makes contact with the relatively conserved extended −10 motif (Fig 2C) via side chains of R91 (Fig 2A and D), which is consistent with the previous report (Koo et al, 2009). The core −10 element C−13G−12ATAAN−7 is conserved in promoters of σ28-dependent flagellar genes (Fig 2C and Appendix Table S2) (Yu et al, 2006a; Zhao et al, 2007; Fitzgerald, 2014), and the major difference with that of σ70 is the −13C and −12G, which have been determined to be essential for σ28-dependent promoters (Yu et al, 2006a; Koo et al, 2009). Consistently, sequence alignment shows that residues with direct contact to these two base pairs in σ28 (R58, Q73, R74, R95, and R98) are different from those in σ70 and σ38 (Fig EV3A). Figure 2. Promoter recognition by the σ28 factor Summary of protein–nucleic acid interactions. Solid and dashed lines represent polar interactions and hydrogen bonds, respectively. The −35 and −10 elements are colored in red letters. The extended −10 motif is in magenta. Recognition of the promoter −10 element. The RNAP is shown in surface representation: β subunit, cyan; β′ subunit, pink; β′ lid, green; β′ rubber, yellow; and σ28, wheat. The right and left side insets at the bottom part are the zoom-in views of the interactions on the NT-strand and T-strand, respectively. Conserved sequences around the −10 element of flagellar promoters (from −16 to −7 positions) generated by WebLogo (Crooks et al, 2004). Promoter sequences used in this analysis are shown in Appendix Table S2. Recognition of the promoter extended −10 element. Recognition of the promoter −35 element by the σ4 domain. Influence of mutating σ4 on promoter recognition. Data Information: In (F), quantifications are mean ± SD from three determinations. Statistical analyses were performed using the unpaired Student's t-test (two-tailed). **P < 0.01. Source data are available online for this figure. Source Data for Figure 2 [embj2020104389-sup-0003-SDataFig2.zip] Download figure Download PowerPoint Apparently, the interactions between σ28 and −35 element are not as extensive as the recognition on −10 element. Being different from previously predicted T-34AAAGTTT-27 sequence (Yu et al, 2006b), our σ28-TIC structures show that A-36ATAAAG-30, which is corresponding to the position of −35 element determined in σ70-TIC structures (Appendix Fig S1), should be the −35 element region recognized by σ28 (Fig 2A and E). Two positively charged residues recognize the phosphate groups of the −35 element: K208/−30C (T-strand), K208/−31T (T-strand), and R220/−36A (NT-strand) (Fig 2A and E). Mutation of the two residues (K208A-R220A) significantly decreased the transcriptional activity of σ28-RNAP holoenzyme on tarp (Fig 2F). The number of residues recognizing −35 element in σ28 is obviously less than that in σ70 (Campbell et al, 2002), indicating that the interaction might be less extensive than that for σ70. The first round of focused classification and refinement also showed that only 33.33% of the particles have clear densities on −35 element region (Fig EV1D), suggesting a weak recognition or a less frequently occupied conformation. In addition, on the basis of the comparison with the σ38-TIC structure (PDB ID 5IPL) and σ70-TIC structure (PDB ID 4YLN), the position of σ4 helices in our structure is similar to that in the σ38-TIC, while they are around 2–3 Å farther away from the −35 element than the σ4 helices in σ70-TIC (Appendix Fig S1), indicating that the recognition on the −35 element in the σ28-TIC is weaker than that in the σ70-TIC. Recognition of the upstream NT-strand DNA in the spacer by β′ ZBD Remarkably, β′ ZBD of RNAP adopts an extended conformation in the state 2 structure compared with that in the state 1 structure (Fig 1C). The β′ ZBD locates at the N-terminal of β′ subunit, and one zinc ion is coordinated by four cysteines of ZBD (Fig 3A). The β′ ZBD in the state 1 architecture is around 12 Å away from the upstream DNA and makes contact with the σ4 domain; in the state 2 structure, ZBD is dissociated from the σ4 domain with a shift of ~9 Å and reaches to the upstream NT-strand DNA (Figs 1C, 3A, and Appendix Fig S2A). In the σ38-TIC structure (PDB ID 5IPL) and σ70-TIC structure (PDB ID 4YLN), β′ ZBDs situate at similar positions as observed in the state 1 structure. Superimposition of them with our state 2 structure also shows ~10 Å distance shift of Zn2+ (Appendix Fig S2B and C). The interface on β′ ZBD possesses a patch of positive charges (Appendix Fig S2D). Together with the highly positively charged regions of the σ2, σ3, and σ4 domains (Appendix Fig S2D), we propose that interactions on β′ ZBD might contribute to a more stable promoter binding and more efficient transcription initiation. Figure 3. Role of β′ ZBD in activating σ28-dependent transcription A. Specific interactions between the upstream DNA (−25 and −26 nucleotides) and β′ ZBD. B, C. Effects of deleting β′ ZBD on transcriptional activities of RNAP on different promoters. The fliCp (B) was used as template for σ28-dependent transcription, and the lacUV5p (C) was used in σ70-dependent transcription. RNA products indicated by solid triangles were quantified, and the signal obtained from 100 nM wild-type RNAP was normalized to 1 in each test, respectively. D. Transcriptional activities of ZBD-mutated σ28-RNAP on fliCp. RNA products indicated by solid triangles were quantified. E. Binding of σ28-RNAP with fliCp as detected by EMSA (left). The heparin-resistant RNAP–promoter open complex is indicated as “RPo”, and the heparin-sensitive RNAP–promoter closed complex is shown as “RPc”. Percentages of RNAP–promoter open complex (RPo) and closed complex (RPc) in EMSA were performed using σ28-RNAP and fliCp (right). F. DNase I footprinting of the fliCp complex with σ28-RNAP. Promoter DNA was labeled on the non-template strand. The promoter region protected by RNAP is indicated by a red box at the bottom panel. Data Information: In (B–E), data are mean ± SD from three determinations. Individual values of replicates are shown as dots. Statistical analyses were performed using the unpaired Student's t-test (two-tailed). *P < 0.05, **P < 0.01. Source data are available online for this figure. Source Data for Figure 3 [embj2020104389-sup-0004-SDataFig3.zip] Download figure Download PowerPoint Consistent with our hypothesis, the ZBD-deleted RNAP (ΔZBD) showed reduced transcriptional activity when reconstituted with σ28 on tarp (Fig EV4A and B) or fliCp promoter (Kundu et al, 1997) (Figs 3B and EV4C), but did not decrease activity with the σ70 on the classical lacUV5p (Figs 3C and EV4D), suggesting a specific role of β′ ZBD. Moreover, the ZBD interacts with the NT-strand mainly through polar interactions: K74/−26C, K74/−25C, and K87/−26C (Fig 3A). Single mutation of either K74 or K87 (named as K74A and K87A, respectively) or both residues (named as K74A–K87A), or mutation of four cysteines in β′ ZBD (named as 4CS) in the RNAP core enzyme, all showed decreased transcriptional efficiency when reconstituted with σ28 (Figs 3D, and EV4E–G), but not with σ70 on lacUV5p (Fig EV4H). Additionally, the ZBD-mutated RNAPs showed decreased promoter binding on fliCp when reconstituted with σ28 (Fig 3E and F), but did not significantly influence promoter binding with the σ70 on lacUV5p (Fig EV4I and J). Importantly, the ZBD-mutated RNAPs formed a higher ratio of unstable heparin-sensitive “closed complex” and a lower ratio of heparin-resistant “open complex” for σ28 (Fig 3E). Consistently, DNase I footprinting assay showed that σ28-WT RNAP protected the fliCp from +16 to −41 positions (Fig 3F), a representative protected region for RPo complex (Chen et al, 2020), but σ28 reconstituted with ΔZBD showed a shorter protected region from +6 to −39 (Fig EV4K), which is characteristic of intermediates between RPc and RPo (Chen et al, 2020). Together, these in vitro data reveal that β′ ZBD significantly contributes to promoter binding of σ28-RNAP and promotes the formation of heparin-stable complexes, some of which are RPo. Click here to expand this figure. Figure EV4. Role of β′ ZBD in promoter binding and transcription A–G. Full image of transcripts from wild-type (WT) or ZBD-mutated RNAPs on different promoters. The amount of abortive transcripts showed a similar trend as runoff products for each sample. The tarp (A, B) and fliCp (C) were used as templates for σ28-dependent transcription, the lacUV5p (D) was used in σ70-dependent transcription, and the fliCp (E) and tarp (F, G) were used as templates for transcription of ZBD point-mutant RNAPs. The signal obtained from 100 nM wild-type RNAP was normalized to 1 in each test, respectively. Data are mean ± SD from three determinations. Individual values of replicates are shown as dots. Statistical analyses were performed using the unpaired Student's t-test (two-tailed). *P < 0.05, **P < 0.01. H. Effects of deleting β′ ZBD on transcriptional activities of σ70-RNAP on lacUV5p. I. Bindings of σ70-RNAP with lacUV5p as detected by EMSA. J. DNase I footprinting of the lacUV5p complex with σ70-RNAP. The promoter region protected by RNAP is indicated by red box at the bottom panel. K. DNase I footprinting of the fliCp complex with σ28-RNAP-ΔZBD. The promoter region protected by σ28-RNAP-ΔZBD is indicated by red box, and regions showing reduced protection by σ28-RNAP-ΔZBD compared with w" @default.
• W3031290989 created "2020-06-05" @default.
• W3031290989 creator A5005433211 @default.
• W3031290989 creator A5006923052 @default.
• W3031290989 creator A5028043271 @default.
• W3031290989 creator A5030980350 @default.
• W3031290989 creator A5036961309 @default.
• W3031290989 creator A5043398732 @default.
• W3031290989 creator A5059637663 @default.
• W3031290989 creator A5084070199 @default.
• W3031290989 date "2020-06-02" @default.
• W3031290989 modified "2023-10-10" @default.
• W3031290989 title "Structural basis of bacterial σ ²⁸ ‐mediated transcription reveals roles of the <scp>RNA</scp> polymerase zinc‐binding domain" @default.
• W3031290989 cites W1505365127 @default.
• W3031290989 cites W1543575219 @default.
• W3031290989 cites W1551727602 @default.
• W3031290989 cites W1568431853 @default.
• W3031290989 cites W1573111727 @default.
• W3031290989 cites W1834065866 @default.
• W3031290989 cites W1912050508 @default.
• W3031290989 cites W1913223174 @default.
• W3031290989 cites W1959539540 @default.
• W3031290989 cites W1963792029 @default.
• W3031290989 cites W1967048697 @default.
• W3031290989 cites W1974956446 @default.
• W3031290989 cites W1981774490 @default.
• W3031290989 cites W1985975342 @default.
• W3031290989 cites W1987267671 @default.
• W3031290989 cites W1989380134 @default.
• W3031290989 cites W1991640026 @default.
• W3031290989 cites W1999434982 @default.
• W3031290989 cites W2013831456 @default.
• W3031290989 cites W2034287170 @default.
• W3031290989 cites W2034373809 @default.
• W3031290989 cites W2035607033 @default.
• W3031290989 cites W2038203831 @default.
• W3031290989 cites W2046831618 @default.
• W3031290989 cites W2048323317 @default.
• W3031290989 cites W2051433813 @default.
• W3031290989 cites W2052094781 @default.
• W3031290989 cites W2052660926 @default.
• W3031290989 cites W2054721713 @default.
• W3031290989 cites W2054770020 @default.
• W3031290989 cites W2055506322 @default.
• W3031290989 cites W2072021660 @default.
• W3031290989 cites W2075716499 @default.
• W3031290989 cites W2079224194 @default.
• W3031290989 cites W2100869077 @default.
• W3031290989 cites W2104088896 @default.
• W3031290989 cites W2111441093 @default.
• W3031290989 cites W2119001327 @default.
• W3031290989 cites W2120263648 @default.
• W3031290989 cites W2123402293 @default.
• W3031290989 cites W2130512803 @default.
• W3031290989 cites W2132629607 @default.
• W3031290989 cites W2134226269 @default.
• W3031290989 cites W2136628260 @default.
• W3031290989 cites W2140803417 @default.
• W3031290989 cites W2143641256 @default.
• W3031290989 cites W2144081223 @default.
• W3031290989 cites W2146424568 @default.
• W3031290989 cites W2146470614 @default.
• W3031290989 cites W2148941629 @default.
• W3031290989 cites W2152066012 @default.
• W3031290989 cites W2154714625 @default.
• W3031290989 cites W2158266834 @default.
• W3031290989 cites W2158573520 @default.
• W3031290989 cites W2160084442 @default.
• W3031290989 cites W2160898059 @default.
• W3031290989 cites W2169131251 @default.
• W3031290989 cites W2174270021 @default.
• W3031290989 cites W2180229411 @default.
• W3031290989 cites W2294658832 @default.
• W3031290989 cites W2315845140 @default.
• W3031290989 cites W2411563451 @default.
• W3031290989 cites W2516060948 @default.
• W3031290989 cites W2569618521 @default.
• W3031290989 cites W2735563253 @default.
• W3031290989 cites W2742975915 @default.
• W3031290989 cites W2769015804 @default.
• W3031290989 cites W2786623728 @default.
• W3031290989 cites W2791354025 @default.
• W3031290989 cites W2791670278 @default.
• W3031290989 cites W2883041598 @default.
• W3031290989 cites W2910191461 @default.
• W3031290989 cites W2911917902 @default.
• W3031290989 cites W2916993760 @default.
• W3031290989 cites W2953320394 @default.
• W3031290989 cites W3012214096 @default.
• W3031290989 doi "https://doi.org/10.15252/embj.2020104389" @default.
• W3031290989 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7360974" @default.
• W3031290989 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32484956" @default.
• W3031290989 hasPublicationYear "2020" @default.
• W3031290989 type Work @default.
• W3031290989 sameAs 3031290989 @default.
• W3031290989 citedByCount "17" @default.
• W3031290989 countsByYear W30312909892021 @default.
• W3031290989 countsByYear W30312909892022 @default.
• W3031290989 countsByYear W30312909892023 @default.
• W3031290989 crossrefType "journal-article" @default.

• next