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Structural insight into the transcription activation mechanism of the phage
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Mor/C family activators
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Jing Shi1,#,*, Zonghang Ye1,#, Yirong Huang1,#, Liqiao Xu2,#, Simin Xu1, Lihong Xie3,4,5, Wei
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Chen6, Lu Wang1, Zhenzhen Feng1, Qian Song1, Shuang Wang7,*, Yu Feng2,*, Wei Lin1,8,*
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1 School of Medicine, Nanjing University of Chinese Medicine, Department of Infectious
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Diseases, Nanjing Drum Tower Hospital, Nanjing 210023, China
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2 Department of Biophysics, and Department of Infectious Disease of Sir Run Run Shaw
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Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
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3 MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of
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Biophotonics, South China Normal University, Guangzhou 510631, China.
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4 Guangdong Key Laboratory of Laser Life Science, Colle Zhejiang University School of
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Medicine ge of Biophotonics, South China Normal University, Guangzhou 510631, China.
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5 Songshan Lake Materials Laboratory, Dongguan 523808, China.
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6 Clinical Research Center, the Second Hospital of Nanjing, Nanjing University of Chinese
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Medicine, Nanjing 211113, China
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7 Chinese Medicine Guangdong Laboratory, Hengqin 519031, China
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8 State Key Laboratory of Bioreactor Engineering, East China University of Science and
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Technology, Shanghai 200032, China
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#, Equal contribution.
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*, Correspondence: shijing301@njucm.edu.cn OR weilin@njucm.edu.cn OR
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yufengjay@zju.edu.cn OR shuangwang@gzucm.edu.cn
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Running title:
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Cryo-EM structures of the phage Mor/C activator-dependent transcription activation complexes
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ABSTRACT
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Bacteriophage Mu, a temperate phage that infects E. coli K-12 and other enteric bacteria,
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precisely controls its replication cycle through hijacking host RNA polymerase (RNAP) by the
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middle operon regulator Mor and the late gene transcription activator C. Though a dimeric
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arrangement and significant conformational changes are proposed for the distinct Mor/C family
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activators, the underlying transcription activation mechanism remains unclear. In this study, we
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present two cryo-EM structures of the transcription activation complex (Mor-TAC and C-TAC)
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with phage Mu middle and late gene promoters, respectively. Remarkably, the Mor/C activators
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bind to promoter DNA as a centrosymmetric tetramer rather than as the proposed dimer,
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concurrently stabilizing by the N-terminal dimerization domains and C-termini. The C-terminal
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DNA binding domains and two anti-β-strands simultaneously interact with two adjacent DNA
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major grooves. The activators also engage a variety of interactions with the conserved domains
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(αCTD, σ70R4, and β FTH) of RNAP, providing evidences for a recruitment mechanism. In
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addition, single-molecule FRET assays show that C significantly enhances RPitc formation,
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suggesting a different multi-step activation mechanism for C. Collectively, these findings reveal
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the unique transcription activation mechanism of tetrameric Mor/C family activators,
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unraveling a novel mode of phage hijacking and bacterial transcription regulation.
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Keywords: phage hijacking, RNA polymerase, transcription activator, transcriptional
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activation complexes, centrosymmetric tetramer
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INTRODUCTION
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Though encompassing simple regulatory systems, the ubiquitously distributed phages
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possess the capability to resist bacterial adversities and govern phage replication cycles (1).
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They primarily achieve this by encoding various transcription factors that hijack bacterial
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transcriptional machineries via intricate protein-DNA and protein-protein interactions during
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the transcription initiation process (2,3). In the model bacteria Escherichia coli (E. coli), the
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multi-subunit RNA polymerase holoenzyme (RNAP, α2ββ'ωσ) is initially assembled by the
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core enzyme and the principal promoter specific factor σ (σ70) (4,5). Accompanied by
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interacting with the β flap and the clamp helices of β' subunit (β' CH), the conserved domains
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R4 (σ70R4) and R2 (σ70R2) of σ70 specifically recognize promoter DNA consensus -35 and -10
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elements, respectively (6). This drives DNA unwinding and isomerizes an RNAP-promoter
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closed complex (RPc) into an RNAP-promoter open complex (RPo) (7-9). With the addition of
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NTPs, RNAP attempts to synthesize nascent RNA short than 12 nucleotides, result in an RNAP-
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promoter initial transcribing complex (RPitc). As RNA length reaches 13–15 nucleotides,
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σ70 disengages from interactions with the promoter DNA and RNAP—a process known as
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promoter escape (or promoter clearance)—thereby transitioning abortive transcription
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initiation into the subsequent transcription elongation stage (10-12). For weak promoters that
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consist of suboptimal promoter elements, various transcription activators join in to compensate
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for instability between the RNAP and promoter DNA, accumulating formation of a
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transcription activation complex (TAC) competent for transcription initiation (13-18).
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Bacteriophage Mu is a temperate phage that infects E. coli K-12 and other enteric bacteria.
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To ensure successful infection, Mu has evolved a complex and elaborate regulatory network to
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control transient expression of distinct phase genes (early, middle, and late genes) and to switch
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on the lytic cycle (19-22). Notably, phage Mu lacks self-encoded RNA polymerase and
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primarily relies on host RNAP and phage-encoded activators to control the weaker promoters
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of middle genes and late genes, making Mu a valuable model system for studying phage
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hijacking strategies and bacterial transcriptional regulation. The middle operon regulator (Mor)
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activates transcription of the middle gene promoter (Pm) which controls the expression of the
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late transcription activator C (22-25). While C activates transcription of four late promoters
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(Pmom, Plys, PI, PP) that regulate phage DNA modification, morphogenesis, and cell lysis
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(20,21,26-29). Both structural and bioinformatics analyses reveal that Mor and C exhibit
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significant structural similarities, including an acidic N-terminal dimerization domain (DD), a
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conserved 12-amino acid β-strand linker, and a basic C-terminal DNA binding domain (DBD)
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(23,30-35)(Fig. S1). Interestingly, the Mor/C proteins, along with the Mu-like phage proteins,
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form a structurally distinct Mor/C family, which has been extensively investigated for decades.
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Yet, the molecular mechanisms underlying transcription activation by these activators remain
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elusive.
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Biochemical assays have shown that both Mor and C proteins form dimers in solution,
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which may enable dimeric binding to the imperfect dyad-symmetry elements in middle and late
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genes of phage Mu, further recruiting RNAP to promoter DNA (23,30,35,36). While the crystal
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structure and mutational analyses of Mor have highlighted the significance of hydrophobic
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interactions in stabilizing the dimerization domain (DD) and the essential role of the helix-turn-
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helix (HTH) motif in engaging with two adjacent major grooves of DNA, substantial
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conformational changes have also been proposed to address the considerable discrepancies
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observed between the docked model of Mor-DNA and previous chemical cross-linking
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experiments (30,32,36,37). The DNA bending angle induced by Mor binding and the adjacent
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linker, has been suggested to contribute to this conformation change (30,32). Furthermore,
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biochemical and genetic assays have indicated that the C-terminal domain of RNAP α subunit
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(αCTD) and the C-terminal R4 domain of σ70 (σ70R4) are required for Mor-dependent
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transcription of middle genes (25,38-42). Despite the high structural conservation of Mor, it has
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been found that C is critical for the creation of productive TAC, inducing allosteric transitions,
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and enhancing efficient promoter escape via a multistep transcription activation mechanism on
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phage late gene promoters (30,31,43,44). Nonetheless, the lack of structural information
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regarding the binary Mor/C-DNA complex or the ternary Mor/C-DNA-RNAP complex leaves
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long-standing questions. Additionally, the role of the short N-terminus (26 amino acids) and C-
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terminus (9 amino acids) unresolved in the crystal structure remains an intriguing query that
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warrants further exploration.
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In the present work, we report the cryo-EM structures of phage Mu Mor-dependent
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transcriptional activation complex (Mor-TAC, 4.12 Å) and C-dependent transcriptional
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activation complex (C-TAC, 3.14 Å), which are assembled on phage Mu middle and late gene
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promoters, respectively. Strikingly, in both Mor-TAC and C-TAC, the Mor and C proteins bind
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to promoter DNA as a centrosymmetric tetramer, with the N-terminal DDs and C-termini
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facilitating this unique architecture. The C-terminal DNA binding domains and two anti-β-
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strands concurrently interact with the two adjacent DNA major grooves upstream of promoter
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-35 element, while two flanking helices α2 make contacts with the bilateral DNA minor grooves.
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Furthermore, the stability of TACs is further stabilized through diverse interactions between the
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Mor/C activators and the conserved domains (αCTD, σ70R4, and β FTH) of RNAP, providing
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structural evidences for the recruitment mechanisms for Mor and C. Consistently, mutagenesis
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and single-molecule FRET assays proved C significantly enhances RPitc formation, thus
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supporting a different multi-step transcription activation mechanism for C. Altogether, these
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data offer molecular insights into the unique transcription activation mechanisms of tetrameric
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Mor/C family activators, unraveling a novel mode of phage hijacking and bacterial transcription
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regulation.
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MATERIAL AND METHODS
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Preparation of plasmids and DNA
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Plasmids of pET28a-mor carrying N-terminal 6*His tagged phage Mu middle gene
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activator Mor encoding gene mor and pET28a-c carrying N-terminal 6*His tagged phage Mu
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late gene activator C encoding gene c, both under the control of T7 promoter, were synthesized
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by Sangon Biotech, Inc. The Pm DNA fragment harboring the phage Mu middle gene promoter
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Pm (-87 to +30, including Mor binding box) fused with a following RNA aptamer mango III
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encoding sequence mango (45,46) was amplified by de novo PCR by using primers of Pm_F,
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Pm_F1, Pm_R1, and mango_R (Table S1). Similarly, the Pmom DNA fragment harboring the
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phage Mu late gene promoter Pm (-81 to +30, including C binding box) fused with a following
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mango sequence was prepared by using primers of mom_F, mom _F1, mom_R1, and mango_R
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(Table S2). Plasmids of pGEX-4T-Mor and pGEX-4T-C were constructed by amplifying target
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genes and pGEX-4T-1 vector fragment using the corresponding primers (Tables S1 and S2)
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and through homologous recombination methods according to the manual instructions
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(Vazyme, Inc). All of the other primers used for constructing mutants of Mor and C are listed
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in Tables S1 and S2.
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Purification of Phage Mu Mor and C
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E. coli phage Mu late gene activator C was transformed with the plasmid pET28a-c or
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pET28a-c derivatives. The identified positive transformants were firstly inoculated at 37 °C
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until OD600 reached 0.8, and induced with 0.5 mM IPTG at 18 °C for 16 h. The cells were then
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harvested, resuspended, and lysed in buffer A (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5%
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glycerol). The supernatant centrifuged at 12800 g for 30 min was loaded onto a 3 ml Ni-NTA
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agarose column (Qiagen, Inc.) equilibrated with buffer A. The column was sequentially washed
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with 15 ml buffer A containing 25 mM, 40 mM imidazole and eluted with 15ml buffer A
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containing 200 mM imidazole. The targeted fractions containing Mor were verified by SDS-
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PAGE and concentrated for further analysis. The derivatives of C were purified and stored as
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the wild type (WT) proteins. By analogy, the GST-C was loaded and eluted from the glutathione
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agarose resin column and further purified to high homogeneity by a HiLoad 16/600 Superdex
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75 column (GE Healthcare, Inc.) in buffer B (20 mM Tris-HCl, pH 8.0, 75 mM NaCl, 5 mM
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MgCl2, 1 mM DTT).
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Likewise, E. coli phage Mu middle gene activator Mor was transformed with the plasmid
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pET28a-mor or pET28a-mor derivatives. Then the Mor proteins and its derivatives were
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prepared to high purity through similar affinity chromatography columns and gel filtration
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chromatography column as described above for C, which allows for subsequent experimental
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analysis.
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Purification of E. coli RNAP
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E. coli RNAP was prepared from E. coli strain BL21(DE3) (Invitrogen, Inc.) transformed
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with plasmids of pGEMD (47) and pIA900 (48) by following the methods as described
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previously (49,50) and indicated in the present work. Single positive colonies were used to
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incubate, amplify, and induce protein expression by addition of 0.5 mM IPTG at 18 °C for 16
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h. After harvest by centrifugation (6000 g; 15 min at 4 C), the cell pellets were resuspended
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and lysed in 20 ml lysis buffer A according to per liter of LB medium. Then the supernatant
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centrifuged was sequentially processed through Polyethylenimine (PEI) precipitation (w/v,
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0.7%), washing with buffer C (20 mM Tris–HCl pH 7.9 and 5% glycerol) containing 500 mM
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NaCl for three times, extraction with buffer C containing 1000 mM NaCl, ammonium sulfate
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precipitation (w/v, 30%), resuspending with buffer C containing 500 mM NaCl, and loading
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onto a pre-equilibrated Ni-NTA agarose (Qiagen, Inc.) column. After washing with buffer C
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containing 10 mM imidazole, the column eluted with buffer C containing 200 mM imidazole.
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Subsequently, the eluate was further purified through a Mono Q 10/100 GL column (GE
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Healthcare, Inc.) with a 160 ml linear gradient of 300-500 mM NaCl in buffer D (20 mM Tris-
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HCl, pH 7.9, 5% glycerol, 1 mM EDTA and 1 mM DTT). Target fractions carrying E. coli
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RNAP were applied and purified to high homogeneity by a HiLoad 16/600 Superdex 200
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column (GE Healthcare, Inc.) in buffer B. Fractions were pooled and concentrated to 7.6 mg/ml.
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Assembly of phage Mu Mor–TAC and C-TAC
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The template strand DNA (T) and nontemplate strand DNA (NT) of Pm scaffold and Pmom
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scaffold were synthesized by Sangon Biotech, Inc, and annealed at 1:1 ratio in 10 mM Tris–
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HCl, pH 7.9, 0.2 M NaCl. Then, the assembly of the C-TAC or Mor-TAC was initiated by
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incubating E. coli RNAP, Pmom scaffold (or Pm scaffold), and phage Mu C protein (or Mor
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protein) in a molar ratio of 1: 1.1: 8 at 37 °C for 10 min, and then at 4 °C overnight. After
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centrifugation, the resultant supernatant was loaded onto a HiLoad 16/600 Superdex 200
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column (GE Healthcare, Inc.) equilibrated in buffer B, and the column was eluted with 120 mL
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of the same buffer. Following verification via SDS-PAGE, the target fractions containing each
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component of the assembled C-TAC or Mor-TAC were concentrated to 20.6 mg/ml (24.5 mg/ml)
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by using Amicon Ultra centrifugal filters (10 kDa MWCO, Merck Millipore, Inc.). The
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Oligonucleotides synthesized for the preparation of the DNA scaffolds are detailed in Tables
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S1 and S2.
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Cryo-EM grid preparation
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Initially, the Quantifoil grids (R1.2/1.3 Cu 300 mesh holey carbon grids; Quantifoil, Inc.)
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underwent glow_discharge for 120 seconds at a current of at 15 mA. Following incubation with
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6 mM CHAPSO (Hampton Research Inc.) for 1 min at 25 ℃, 3 μL of the phage Mu C-TAC
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(or Mor-TAC) sample was loaded onto the grids. After blotting with Vitrobot Mark IV (FEI),
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the grids were immediately plunge-frozen in liquid ethane with 95 % chamber humidity at 10 ℃.
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Finally, grids exhibiting a moderate density and uniform distribution of single particles were
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selected for extensive cryo-EM data collection.
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Cryo-EM data collection and processing
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Cryo-EM data for C or Mor-dependent transcription activation complex was acquired
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using a consistent set of parameters on a 300 kV Titan Krios (FEI, Inc.) equipped with a K3
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Summit direct electron detector. The data were processed sequentially with the appropriate
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cryo-EM data analysis software including CryoSPARC v4.2 (51) (Table 1, and Figs. S2, S3).
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A varying number of images were recorded using the EPU software in counting or super
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resolution mode, featuring a pixel size of 1.2 Å or 1.1 Å for C-TAC or Mor-TAC, a dose rate
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of 10 e/pixel/s, and an electron exposure dose of 50 e/Å2. Movies were captured over a duration
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of 8.38 seconds with the defocus range varying from -2.0 μm to -1.0 μm. Subframes of
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individual movies were aligned using MotionCor2 (52), While the contrast-transfer-function
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for each summed image was estimated using CTFFIND4 (53). From the summed images,
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particles were picked by blob picker, template picker and subjected to iterative 2D classification
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in CryoSPARC v4.2. The resulting 2D classes, exhibiting diverse orientations were further
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selected, manually inspected, and subjected to ab-initio reconstruction and hetero refinement.
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By eliminating poorly populated classes, the selected particles were then subjected with or
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without masked 3D classification focusing on upstream DNA region of C-TAC or Mor-TAC.
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Subsequently, particles from the optimal class, which displayed clear density for RNAP, DNA,
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C or Mor were re-processed through homogeneous refinement, non-uniform refinement, CTF-
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refinement, local resolution estimation, and local filtering, ultimately yielding the final map in
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CryoSPARC v4.2. The final particles were further subjected to particle subtraction to preserve
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the signal from the upstream C or Mor binding regions, followed by masked local refinements
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to improve the map quality and interpretability.
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The final mean map resolutions for C or Mor-dependent transcription activation complex,
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as assessed using the Gold-standard Fourier-shell-correlation method, are as follows: 3.14 Å
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for C-TAC, 4.12 Å for Mor-TAC, 3.97 Å for the C region in C-TAC, and 6.92 Å for the Mor
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region in Mor-TAC (Table 1 and Figs. S4, S5).
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Cryo-EM model building and refinement
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The model of RNAP and the downstream DNA, derived from the cryo-EM structure of
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E.coli RPo (PDB ID: 6OUL), as well as the predicted structure of C (ID: AF-E0J3R8-F1) or
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crystal structure of Mor (PDB ID: 1RR7) were fitted into the cryo-EM density map of C-TAC
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and Mor-TAC using Chimera (54) to generate a preliminary structural model. The model of the
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upstream nucleic acids was built manually using Coot (55). The coordinates were subsequently
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calculated and validated through real-space refinement incorporating secondary structure
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restraints in Coot and Phenix (v1.19.2) (56). Structures were analyzed using Chimera and
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PyMOL(57). The Map versus Model FSCs of the four cryo-EM maps (C-TAC, Mor-TAC) in
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this work were generated by Phenix. The statistics of cryo-EM refinement were summarized in
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Table 1.
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In vitro transcription assay
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To assess the effects of Mor or C on activating transcription of the phage Mu middle gene
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promoter Pm or late gene promoter Pmom, we performed in vitro transcription assays with a
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96-well microplate in the transcription buffer (20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 5%
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glycerol, 10 mM MgCl2). Each component was included in the reaction mixture (80 μl) as
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following: 0 or 4 μM C or C mutants (Mor or Mor mutants), 0.1 μM RNAP, 50 nM Pmom DNA
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(or Pm DNA), 0.1 mM NTP mix (ATP, UTP, GTP, and CTP), and 1 μM TO1-Biotin. Initially,
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RNAP, promoter DNA, and C or C mutants (Mor or Mor mutants) were incubated for 15 min
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at 37 ℃. Then, NTP mix and TO1-biotin were supplemented into the mixture to trigger
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transcription initiation for 10 min at 37 ℃. Finally, the fluorescence emission intensities were
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measured using a multimode plate reader (EnVision, PerkinElmer Inc) with an excitation
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wavelength at 510 nm and an emission wavelength at 535 nm. Relative transcription activities
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of the relevant derivatives were calculated using the following formula (1):
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A = (I − I0) / (IWT − I0) (1)
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where IWT and I are the fluorescence intensities in the presence of C or C mutants (Mor or
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Mor mutants), I0 is the fluorescence intensity in the absence of C (or Mor) protein.
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Electrophoretic mobility shift assay
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Electrophoretic mobility shift assays (EMSA) of phage Mu C (or Mor) protein were
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performed in reaction mixtures (20 μl) containing: 30 nM Pmom DNA (or Pm DNA), 0 or 0.15
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μM E. coli RNAP, and supplemented with 4 μM GST-C (or GST-Mor) in the EMSA buffer (20
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mM Tris–HCl, pH8.0, 50 mM NaCl, 5 mM MgCl2, 5% glycerol). Firstly, the promoter DNA,
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E. coli RNAP were incubated for 10 min at 30 ℃. Then the GST-C (or GST-Mor) protein was
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added into the reaction mixture, and incubated for another 20 min at 30 ℃ before addition of
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0.075 mg/ml heparin. Finally, the samples were applied to 5% polyacrylamide slab gels (29:1
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acrylamide/bisacrylamide), electrophoresed in 90 mM Tris borate, pH 8.0, and 20 mM EDTA,
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and stained with 4S Red Plus Nucleic Acid Stain (Sangon Biotech, Inc.) according to the
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procedure of the manufacturer.
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Single-molecule FRET assay
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HPLC purified DNA oligos (C-nonTem/C-Tem, and Mor-nonTem/Mor-Tem listed in Tables
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S1 and S2, the underlined “T” is labeled with Cy3 for non-template strand and Cy5 for template
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strand, respectively, Sangon Biotech) were annealed at 1:1 molar ratio in the transcription buffer by
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heating to 95 °C for 5 min followed by slowly cooling down to room temperature in about two hours,
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named Pmom-DNA and Pm-DNA, respectively. After adding a final concentration of 0.5 mg/ml
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BSA, the annealed DNA constructs was aliquoted and stored at -20 °C.
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In single-molecule FRET assay, the Cy3/Cy5 doubly labeled DNA substrates were tethered to
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the PEG surface of a flow chamber precoated with streptavidin (ThermoFisher Scientific).
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Experiments with DNA alone or in presence of 10 nM RNAP, or 10 nM RNAP + 100 µM NTPs, or
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10 nM RNAP + 100 µM NTPs + 500 nM C protein or Mor protein, were performed in the
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transcription buffer containing 0.5 mg/ml BSA, 2.5 mM PCA (protocatechuic acid, Sigma-Aldrich),
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50 nM PCD (protocatechuate-3,4-dioxygenase, Sigma-Aldrich) and 1 mM Trolox (Sigma-Aldrich).
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For the experiments with C or Mor proteins, 500 nM C or Mor proteins were incubated with surface-
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tethered DNA for 10 min at room temperature, respectively, followed by addition of 10 nM RNAP
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+ 100 µM NTPs + 500 nM C protein or Mor protein in the transcription buffer containing 0.5 mg/ml
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BSA, 2.5 mM PCA (protocatechuic acid, Sigma-Aldrich), 50 nM PCD (protocatechuate-3,4-
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dioxygenase, Sigma-Aldrich) and 1 mM Trolox (Sigma-Aldrich). Data was collected at an
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acquisition rate of 10 Hz on a home-made TIRF-based microscope under the excitation of 532 nm
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laser (Coherent). Fluorescence was imaged by an objective (Apo TIRF 100x, 1.49NA, Olympus),
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splitted into two channels and then collected on an EMCCD (iXon Ultra 897, Andor). Raw data was
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extracted, and background corrected. The FRET value or the number of transcription events (RPo,
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RPitc or none) were analyzed manually.
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RESULTS
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Overall structures of Mor-TAC and C-TAC
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To elucidate the structural basis underlying the transcription activation mechanism of the
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distinct Mor/C family factors, we purified E. coli RNAP, and phage Mu Mor/C proteins to high
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homogeneity, and prepared the phage Mu middle gene promoter Pm scaffold and the late gene
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promoter Pmom scaffold. Each of the scaffold contains a consensus –10 element, a transcription
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bubble, and an imperfect dyad-symmetry element (comprising A site and B site) located just
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upstream of the nonoptimal -35 element (Fig. 1A and Fig. S4). In vitro transcription assays
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demonstrated that Mor and C activated transcription of the phage Mu middle gene promoter
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Pm and late gene promoter Pmom, respectively (Fig. S5A). Electrophoretic mobility shift
316
assays (EMSA) yielded shifted bands for Mor-TAC and C-TAC compared to the binary Mor/C-
317
DNA complex or the RNAP-DNA complex (Fig. S5B). These findings are in good agreement
318
with the previous observations that Mor recruits RNAP to Pm rather than repositioning a
319
prebound RNAP, a process that occurs concurrently for C-dependent repositioning of RNAP at
320
Pmom (39,43). Consistently, gel filtration maps and SDS-PAGE analysis of the purified phage
321
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12
Mu Mor-TAC and C-TAC demonstrated the proportional appearance of each protein
322
component (Fig. S5, C and D).
323
After data collection of the optimized TACs samples, we processed the cryo-EM data and
324
performed model building by combination of different structural software. The cryo-EM maps
325
of Mor-TAC and C-TAC were determined at nominal resolutions of 4.12 Å and 3.14 Å,
326
respectively (Fig. 1, Tables 1, 2, and Figs. S2-S4, S6, S7). The RNAP subunits and the
327
downstream DNA from the cryo-EM structure model of E. coli RPo (PDB ID: 6OUL) were
328
able to be fitted into the discerned electron densities of the RNAP core regions in TACs (58).
329
Additionally, the DDs and DBDs from the reported crystal structures of the Mor dimer (PDB
330
ID: 1RR7) and the AlphaFold-predicted C monomer (ID: AF-E0J3R8-F1) were well fitted into
331
the electron densities surrounding the upstream DNA in Mor-TAC and C-TAC, respectively.
332
Strikingly, the Mor/C proteins bind promoter DNA as centrosymmetric tetramers in both
333
Mor-TAC and C-TAC (Figs. 1, 2, and Figs. S4, S6-S10), which are quite different from the
334
docked dimeric model of Mor and DNA (30,32). In contrast, these differences greatly coincide
335
with the predicted conformation changes for transactivation of the Mor/C proteins. Though the
336
N-terminus and C-terminus are not wholly visualized, the corresponding electron densities from
337
each protomer intertwined together over the tetramer, suggesting positive roles of the termini
338
in stabilizing this unique architecture (Fig. 2, and Figs. S8-S10). Moreover, there are evident
339
overlapping densities between the Mor/C protein and the conserved domains of E. coli RNAP,
340
indicative of extensive interactions between the Mor/C proteins and RNAP (Fig. 3, and Figs.
341
S11, S12), which may facilitate recruitment of RNAP to the promoter region and enhance
342
stabilization of the functional TACs. Besides, the upstream DNA in C-TAC exhibits a larger
343
bending angle compared to that in Mor-TAC or phage λCII-TAC (59), which may provide
344
valuable insights into C-dependent transcription activity on phage Mu late gene promoter (Fig.
345
1, and Fig. S13). Given the higher resolution of C-TAC, more diverse protein-protein
346
interactions, and more complex transactivation mechanism of C compared to Mor, the structural
347
model of C-TAC is emphasized in the following text to elucidate the distinct transcription
348
activation mechanisms of the Mor/C family activators, using Mor-TAC as a comparison.
349
350
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13
Interactions mediate DNA engagement and tetramerization in Mor-TAC and C-TAC
351
In Mor-TAC and C-TAC, both the Mor and C centrosymmetric tetramers undergo
352
significant conformational changes to facilitate DNA engagement and tetramerization through
353
newly uncovered interactions (Fig. 2, and Figs. S2, S6-S10), which align well with predictions
354
from earlier biochemical and crystal structural studies (30,32).
355
In C-TAC, the downstream C dimer, intertwined by CI and CII, and the upstream dimer,
356
associated with CIII and CIV, coordinately assemble into a centrosymmetric tetramer that extends
357
to occupy nearly three helical turns upstream the potential -35 element (Fig. 2A). This
358
positioning coincides with the far apart DNA binding sites (26 bp) observed in previous
359
footprinting assays (30,36). In contrast to the docked Mor-DNA structural model, both the
360
conserved C-terminal HTH motif (comprising residues R98, S110, Q113, Y115, Q116, and
361
R120) of CI constituted by helices α5-α7, and the anti-paralleled β-strands (including residues
362
R72, R73, Y75, P77, and T81) from CIII and CIV move away from the dimerization domain (DD)
363
and insert into the upstream DNA major groove from opposite directions (Fig. 2B). Additionally,
364
two flanking loops connecting helix α2 and helix α3, consisting of residues S29, R30, P32, R33,
365
and S34, make contacts with the DNA backbones of the adjacent DNA minor grooves. By
366
analogy, the centrosymmetric anti-paralleled β-strands from CI and CII and the conserved C-
367
terminal HTH motif of CIII specifically interact the downstream DNA major groove,
368
accompanying by two flanking loops interacting with the adjacent DNA minor grooves (Fig.
369
2B). These interactions allow for stable DNA engagement of the Mor/C proteins at the
370
corresponding binding box, which are further favored by the centrosymmetric tetramer
371
architecture. Except for the clustered hydrophobic and polar interactions involved in each DD
372
of the C dimers (Fig. 2C), which exhibit similarities with those observed in the crystal structure
373
of the Mor dimer, polar contacts between helix α6 of CI and the β-strand of CIV, as well as
374
between helix α6 of CIII and the β-strand of CII contribute to the tetramerization of the CI-CII
375
and CIII-CIV dimers (Fig. 2D), thereby enhancing formation and stability of the centrosymmetric
376
tetramers within the TACs. Notably, mutations of the C residues that are critical for DNA
377
binding and for dimeric and tetrameric interactions compromise the in vitro transcription
378
activities of the C protein (Fig. 2E). Specifically, mutations at residues Y75, Y115, R72, and
379
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14
truncation of the C-terminus (H129term) nearly abolished the relative transcription activity of
380
C, underscoring the essential roles of these residues and the previously uncharacterized C-
381
terminus in sustaining C-dependent transcription activation (30).
382
Similarly, comparable protein-DNA and protein-protein interactions are observed in the
383
structure of Mor-TAC. Mutations affecting these interfaces, as well as truncation of the acid N-
384
terminus (del_N10) or the basic C-terminus (L119term), result in defects in the transcription
385
activities of the Mor protein (Figs. S9, S10), thereby highlighting the significance of these
386
interfaces in promoting Mor-dependent transcription activation.
387
388
Interactions mediate RNAP recruitment in Mor-TAC and C-TAC
389
In addition to the EMSA results which displayed apparent roles in recruiting RNAP (Fig.
390
S5B), structural analysis of both Mor-TAC and C-TAC also provide detailed evidences for
391
RNAP recruitment of the Mor/C proteins (Fig. 3 and Figs. S11, S12).
392
Biochemical and genetic studies have demonstrated that both the middle and late gene
393
promoters of Mu phage contain UP elements (AT-rich element) that may be specifically
394
recognized by the conserved domain of RNAP αCTD (38,40). Mutagenesis experiments also
395
indicate that σ70R4 is required for Mor/C-dependent transcriptional activation (25,42).
396
Supporting these findings, in C-TAC, the conserved domain αCTD of RNAP not only interacts
397
with the UP elements of promoter DNA through its 265 determinant (including residues R265,
398
N268, and N294), but also establishes polar contacts with the C-terminal of helix α7 and the
399
loop connecting helices α5 and α6 from CI, respectively (Fig. 3, A-C). Besides, the residues
400
from the β-strand of CI and helix α3’ of CII interact with the two conserved C-terminal helices
401
of σ70R4 (Fig. 3, D and E), exhibiting a buried surface area of approximately 125 Å2.
402
Accordingly, substitutions of these residues involved suppressed in vitro transcription activity
403
of the C protein, particularly for residues R48, R122, and H129 (Fig. 3F). Likewise, structural
404
analysis and mutagenesis of Mor-TAC yielded comparable results (Fig. S11). Notably, the
405
residue C67 from the CI-CII dimer is likely to make contacts with the residues R599 and I905
406
from the conserved β flap tip helix (βFTH) of RNAP, an interface not observed in Mor-TAC
407
(Fig. S12). These extensive protein-protein interactions involving C may enhance its higher
408
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15
capability to recruit RNAP, thereby facilitating the formation of a more stable C-TAC complex
409
compared to Mor-TAC.
410
411
Single-molecule FRET assays show C significantly promotes RPitc formation
412
To further assess whether C activates transcription initiation in subsequent processes following
413
the formation of TAC as previously proposed, we conducted dynamic single-molecule Förster
414
resonance energy transfer (smFRET) assays by using Mor as a control. We prepared doubly labeled
415
DNA substrates (Cy3 at -4 position of the non-template strand and Cy5 at +17 position of the
416
template strand, Fig. 4A). The FRET between these two dyes was utilized to characterize the
417
changes in their distances during interacting with RNAP, which enable us to monitor the transition
418
states of RNAP during transcription initiation in the presence of either the C protein or the Mor
419
protein (60).
420
As shown in Fig. 4, surface-tethered DNA molecules alone yield a mean FRET value of 0.19
421
(Fig. 4B). This value increases to 0.32 upon the addition of RNAP (Fig. 4C), indicating a distance
422
reduction between the Cy3/Cy5 dyes caused by bending and unwinding of promoter DNA, resulting
423
in the formation of the RPo complex. When RNAP and NTPs are added, 121 out of 263 DNA
424
molecules remain in the RPo state, however, 7 out of these 121 molecules exhibit abrupt FRET
425
increases that exceed those observed in the RPo state, suggesting that RNAP transitions into the
426
initial transcription complex (RPitc) state (Fig. 4D). To investigate the influence of C on the
427
transcription activity of RNAP, C was incubated with the DNA substrate on a PEG surface at room
428
temperature for 10 min, followed by the addition of RNAP, NTPs and C. As anticipated, 307 out of
429
444 molecules formed the RPo state. Notably, 240 of these 307 molecules transitioned into RPitc
430
states, indicating a significant role of C in promoting transcription initiation (Fig. 4E). Consistently,
431
mutations of the relevant C residues (R98A, Y115A, and R122A) resulted in a decreased fraction
432
of RPitc formation (Fig. 4F), indicative of the importance of these residues in activating
433
transcription during the later process. In contrast, similar experiments conducted with the Mor
434
protein on the phage Mu middle gene promoter demonstrated a weaker effect of Mor in facilitating
435
RNAP transcription initiation compared to C (Fig. S14).
436
Collectively, these data suggest that C function as a transcriptional activator in a manner
437
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16
different from Mor during the transcriptional initiation phase after TAC formation, which
438
further supports the previous observations from a dynamic perspective that C also facilitates
439
transcriptional activation by enhancing promoter clearance (43,44).
440
441
DISCUSSION
442
Recently, the emerging cryo-EM single particle reconstruction technology has
443
significantly accelerated structural investigation of phage transcriptional regulation, including
444
large intermediate complexes difficult to determine by X-ray crystallography or NMR (1). Here,
445
by utilizing cryo-EM to investigate the model system phage Mu, we have elucidated long-
446
standing questions regarding the transcription activation mechanisms of the Mor/C family
447
activators through the following new findings.
448
First, the Mor/C family transcription activators form distinct centrosymmetric tetramers
449
that elegantly stabilize Mor-TAC and C-TAC via extensive protein-DNA and protein-protein
450
interactions (Figs. 1, 2 and Figs. S2, S6-S10). Unlike most transcription factors that utilize
451
HTH motifs to engage with the major and minor grooves of DNA, two Mor/C dimers
452
simultaneously interact with two adjacent major grooves concurrently through both their
453
conserved HTH motifs and the unique anti β-strands, accompanying by contacts made through
454
short helices α2 with two bilateral minor grooves. Such protein-DNA interactions are also
455
favored by substantial interactions between Mor/C and the conserved domains (αCTD, σ70R4,
456
and β FTH) of RNAP (Figs. 3, 5A and Figs. S11, S12). Collectively, these interactions lead to
457
significant bending of the upstream promoter DNA in TACs, particularly in C-TAC, compared
458
to the canonical RNA polymerase open complex (RPo) (Fig. 5). These findings, consistent with
459
previous biochemical and genetic assays, provide new detailed evidences for the 'recruitment
460
mechanism' of Mor-dependent transcription activation of phage Mu middle genes, as well as
461
the dual 'recruitment-repositioning mechanisms' for C-dependent transcription activation of
462
phage Mu late genes.
463
Second, significant conformational changes are observed in the homologous Mor-TAC
464
and C-TAC as compared to the crystal structure of the Mor dimer and the docked binary model
465
of Mor and DNA. These changes allow for large occupancy of the Mor/C family activators on
466
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17
promoter DNA, as proposed by biochemical assays (30-32,34,36,44). Unlike other monomeric
467
and dimeric transcription factors, the tetrameric architecture of both Mor and C offers unique
468
advantages: the downstream dimer is essential for interaction with the promoter B site and the
469
conserved domains of RNAP, while the upstream dimer bound to the promoter A site may
470
obstruct additional binding from other transcriptional activators or repressors, thereby
471
significantly enhancing the efficiency of transcription initiation. The centrosymmetric tetramer
472
arrangement differs from the reported tandem GlnR tetramer observed in Mycobacterium
473
tuberculosis GlnR-TAC (61), presenting a novel activation mechanism for transcription factors.
474
Third, the structural, biochemical and kinetic single-molecule FRET assays provide new
475
insights into the distinctions between Mor-TAC and C-TAC. Due to establishing more abundant
476
interactions with the conserved domains (σ70R4 and β FTH) of RNAP, C significantly alters the
477
DNA curvature in the upstream region of C-TAC compared to Mor (Fig. 5A). This alteration is
478
indicated as a prerequisite for σ70R4 binding in the other reported TACs, as well
479
(18,49,59). Though σ70R4 and the suboptimal -35 element of Mor-TAC superimpose well on
480
the structure of RPo-rpsT (PDB ID: 6OUL), the greater DNA bending angle in C-TAC,
481
resulting from the binding of C, extends the distance between σ70R4 and the DNA (Fig. 5B),
482
thereby weakening the reciprocal interactions. This may facilitate subsequent RPitc formation,
483
promoter clearance, and σ escape, which were kinetically verified by single-molecule FRET
484
assays (Fig. 4 and Fig. S14). In contrast to a canonical RPo or TAC with dimeric transcription
485
activators, Mor and C activate transcription of the phage Mu middle and late genes by acting
486
as distinctive centrosymmetric tetramers (Fig. 5C).
487
In contrast to the structure of phage λCII-TAC, this work highlights different intriguing
488
characteristics of the Mor/C family activator-dependent transcription activation: (i) These
489
activators adopt as unique centrosymmetric tetramers and maintain stability of the TACs
490
through more viable and diverse interfaces with both DNA and the conserved domains (αCTD,
491
σ70R4, and β FTH) of RNAP than λCII (Fig. S13). Regarding the DNA binding characteristics
492
as reviewed recently, the Mor/C family activators should be classified as class I transcription
493
factors which bind upstream of promoter -35 element, while the λCII is categorized as a class
494
IV transcription factor that binds both upstream and downstream of promoter -35 element. (ii)
495
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18
The different DNA bending angles observed in Mor-TAC and C-TAC may provide new
496
structural insights into the conformational changes that occur during transcription initiation,
497
reflecting the complexity of transcription regulation in the middle and late genes of phage Mu.
498
(iii) By combing the single molecule FRET assay with the biochemical and structural
499
observations, this study enhances our understanding on the molecular transcription mechanisms
500
of the Mor/C family activators, which elegantly regulate the phage Mu lytic cycle by improving
501
the specificity and efficiency of transcription activation.
502
In summary, our findings reveal the unique transcription activation mechanism of the
503
tetrameric Mor/C family activators, unraveling a novel mode of phage hijacking and bacterial
504
transcription regulation. The key regulatory elements involved in both our and the previous
505
phage TACs may offer new targets to effectively control phage gene expression for specific
506
phage therapy.
507
508
DATA AVAILABILITY
509
The cryo-EM maps and model coordinates have been deposited under the accession
510
numbers: EMDB: 62727 and PDB: 9L0X for the phage Mu Mor-TAC, EMDB: 62728 and PDB:
511
9L0Y for the phage Mu C-TAC. All data are available in the main text or the supplementary
512
materials.
513
514
SUPPLEMENTARY DATA
515
The online version contains supplementary material available at XXX.
516
517
AUTHOR CONTRIBUTIONS
518
J. S., W. L., and S. W. conceived the project. Z.H. Y., Y.R. H., L.X. X., S.M. X., L.H. X.,
519
W. C., Z.Z. F., Q. S., and J. S. prepared the protein samples, performed the biochemical assays,
520
single-molecule FERT assays, and cryo-EM sample assembly. L.Q. X., J. S., and S.M. X
521
prepared cryo-EM samples and data acquisition. W. L. and F. Y. analyzed the cryo-EM data
522
and determined the structures. All authors contributed to data analysis. J. S., W. L., W. C., and
523
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
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19
S. W. wrote the paper with input from all coauthors.
524
525
ACKNOWLEDGEMENTS
526
We appreciate Shenghai Chang at the Center of Cryo-Electron Microscopy in Zhejiang
527
University School of Medicine, Guangyi Li, Liangliang Kong, Jialin Duan, and Yun Song of
528
the Electron Microscopy System at the National Facility for Protein Science in Shanghai
529
(NFPS), Shanghai Advanced Research Institute, Chinese Academy of Sciences, China for
530
providing technical support and assistance in data collection. We thank the Experiment Center
531
for Science and Technology, Nanjing University of Chinese Medicine for experimental
532
assistance. We thank the Core Facilities, Zhejiang University School of Medicine for technical
533
support.
534
535
FUNDING
536
This work was funded by the National Natural Science Foundation of China (32270037,
537
32270192, 82311530689, 82072240, 32471276), the Jiangsu Qinglan Project to J.S., the
538
Natural Science Foundation of Jiangsu Province (BK20211302, SBK2023030145), the
539
National Key R&D Program of China (2023YFC2308200), the landmark talent training project
540
of Nanjing University of Chinese Medicine (RC202404), the special fund project of Nanjing
541
Drum Tower hospital for the transformation of scientific and technological achievements
542
(202404).
543
544
CONFLICT OF INTEREST
545
The authors declare no competing interest.
546
547
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of mom promoter of bacteriophage Mu. J Biol Chem, 281, 8511-8517.
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59. Zhao, M., Gao, B., Wen, A., Feng, Y. and Lu, Y.Q. (2023) Structural basis of lambdaCII-
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60.Koh, H.R., Roy, R., Sorokina, M., Tang, G.Q., Nandakumar, D., Patel, S.S. and Ha, T. (2018)
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al. (2023) Structural insights into the transcription activation mechanism of the global
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The copyright holder for this preprintthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.05.02.651988doi: bioRxiv preprint
24
705
TABLES, FIGURES AND FIGURES LEGENDS
706
707
Tables
708
Table 1. Single particle cryo-EM data collection, processing, and model building for
709
E. coli Mor-TAC and C-TAC.
710
Protein-DNA complex
Mor-TAC
C-TAC
PDB ID
9L0X
9L0Y
EMDB ID
62727
62728
Data collection and processing
Microscope
Titan Krios
Titan Krios
Voltage (kV)
300
300
Detector
K3 summit
K3 summit
Electron exposure (e/Å2)
50
50
Defocus range (m)
-1.0~-2.0
-1.0~-2.0
Data collection mode
super resolution
counting
Physical pixel size (Å/pixel)
1.1
1.2
Symmetry imposed
C1
C1
Initial particle images
412,492
523,383
Final particle images
99,820
217,572
Map resolution (Å)a
4.12
3.14
Refinement
Map sharpening B-factor (Å)
–156
-160
Root-mean-square deviation
Bond length (Å)
0.006
0.003
Bond angle (º)
0.723
0.587
MolProbity statistics
Clashscore
16.13
11.85
Rotamer outliers (%)
0.08
0.25
Coutliers (%)
0.0
0.0
Ramachandran plot
Favored (%)
94.99
96.92
Outliers (%)
0.00
0.00
a Gold-standard FSC 0.143 cutoff criteria.
711
712
713
714
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The copyright holder for this preprintthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.05.02.651988doi: bioRxiv preprint
25
715
716
717
718
Table 2. Single particle cryo-EM data collection, processing for focusing on Mor or C
719
region in E. coli Mor-TAC and C-TAC.
720
721
a Gold-standard FSC 0.143 cutoff criteria.
722
723
724
725
726
727
728
729
730
731
732
733
734
735
Protein-DNA complex
Mor-TAC
C-TAC
EMDB ID
Data collection
63410
63408
Voltage (kV)
300
300
Detector
K3 summit
K3 summit
Electron exposure (e/Å2)
50
50
Defocus range (m)
-1.0~-2.0
-1.0~-2.0
Data collection mode
super resolution
counting
Physical pixel size (Å/pixel)
1.1
1.2
Symmetry imposed
C1
C1
Initial particle images
412,492
523,383
Final particle images
99,820
217,572
Map resolution (Å)a
6.92
3.97
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26
Figures and Figure Legends
736
737
738
Fig. 1 Overall structure of the phage Mu C-TAC.
739
(A) DNA scaffold used in structure determination of Phage Mu C-TAC. (B, C) Two views of
740
the cryo-EM density map (B) and structure model (C) of Phage Mu C-TAC. The EM density
741
maps and cartoon representations of C-TAC are colored as indicated in the color key. Firebrick,
742
NT, non-template-strand promoter DNA; red, T, template-strand promoter DNA. blue, CI;
743
wheat, CII; orange, CIII; Cyan, CIV; light blue, C binding site A; dark blue, C binding site B;
744
yellow, E. coli RNAP σ70 subunit; violet, E. coli RNAP αCTD. The C binding box, -35 element,
745
and -10 element is boxed with yellow, violet, and brown colors, respectively. The EM density
746
maps are colored as indicated in the color key.
747
748
749
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27
750
Fig. 2 C binds promoter DNA as a centrosymmetric tetramer in the phage Mu C-TAC.
751
(A) Relative locations of the centrosymmetric C tetramer at the upstream double-stranded DNA
752
(left panel); structure of C tetramer in cartoon (right panel). (B) The relative locations between
753
CI and CII (left panel) or CIII and CIV (right panel) bound to the promoter DNA. The secondary
754
structural elements involved in C tetramer are labeled, respectively. Colors of CI, CII, CIII, CIV,
755
NT, and T in A-C are shown as in Fig. 1C. (C) Detailed dimerization interactions between CI
756
and CII (left panel) or CIII and CIV (right panel). (D) Detailed tetrameric interactions between CI
757
and CIV or CII and CIII. The secondary structural elements are labeled, respectively. Colors are
758
shown as in Fig. 1. The key residues involved in B-D are shown as spheres in the corresponding
759
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28
colors. (E) Mutations of the C residues involved in DNA binding, dimeric and tetrameric
760
interfaces display reduced transcription activities of the C protein. Data for in vitro transcription
761
assays are means of 3 technical replicates. Error bars represent mean ± SEM of n = 3
762
experiments.
763
764
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29
Fig. 3 C makes extensive interactions with the conserved domains of RNAP in the phage
765
Mu C-TAC.
766
(A) Relative locations of E. coli RNAP αCTD, CI, and the upstream double-stranded DNA. E.
767
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30
coli RNAP αCTD, CI are also represented as magenta or orange cartoon, respectively. RNAP
768
αCTD is also shown in surface. (B) Detailed interactions between E. coli RNAP αCTD and the
769
upstream double-stranded DNA, with key residues involved in αCTD shown as magenta
770
spheres. (C) Detailed interactions between E. coli RNAP αCTD and CI, with key residues
771
involved in αCTD and CI shown as magenta and blue spheres, respectively. (D) Relative
772
locations of E. coli RNAP σ70R4, CI, CII, and the upstream double-stranded DNA. CI, CII and
773
RNAP σAR4 are shown in surface and colored as in Fig.1C. (E) Critical interactions between
774
the CI-CII dimer and E. coli RNAP σ70R4. Residues involved in the magnified interfaces (right
775
panel) between the CI-CII dimer and RNAP σAR4 are shown as yellow (RNAP σAR4), blue (CI),
776
and wheat (CII) spheres, respectively. (F) Mutation of residues of C involved in the above
777
interfaces suppressed in vitro transcription activity. Data for in vitro transcription assays are
778
means of three technical replicates. Error bars represent ± SEM of n = 3 experiments.
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
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31
797
798
799
800
Fig. 4 Characterization of C protein on RNAP transcription activity via single-molecule
801
FRET assay.
802
(A) Schematic of the DNA substrate for the single-molecule FRET assay. Typical trajectories
803
representing the intensities of Cy3 and Cy5 and thus the FRET for each reaction condition (left
804
panels): Surface-tethered DNA alone (B), Surface-tethered DNA + 10 nM RNAP (C), Surface-
805
tethered DNA + 10 nM RNAP + 100 µM NTPs (D), and Surface-tethered DNA + 10 nM RNAP
806
+ 100 µM NTPs + 500 nM C protein (E), respectively. FRET distributions of each condition
807
are fit to a single-Gaussian function yielding peaks at 0.19 ± 0.002 (SEM, N = 166, right panel,
808
B), and 0.32 ± 0.002 (SEM, N = 89, right panel, C). Pie charts representing the fractions of
809
DNA molecules occurred in RPo (N = 114 for D, and 67 for E, yellow), RPitc (the initial
810
transcription complex, N = 7 for D, and 240 for E, green), and without RPo or RPitc complexes
811
(N = 142 for D, and 136 for E, white) in the absence and the presence of C protein, respectively,
812
(right panels). (F) Mutation of the C residues involved in C-DNA, C-αCTD interfaces suppress
813
fractions of RPitc formation.
814
815
816
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The copyright holder for this preprintthis version posted May 6, 2025. ; https://doi.org/10.1101/2025.05.02.651988doi: bioRxiv preprint
32
817
Fig. 5 Proposed transcription activation model for the phage Mu Mor/C family proteins.
818
(A) The interface of σ70R4 with Mor (left panel) or C (middle panel), MorI or CI and σ70R4
819
were represented as surface in wheat, blue and yellow; alignment between the cryo-EM
820
structures of C-TAC and Mor-TAC on the upstream DNA (right panel), the surface
821
representations of MorIII and MorIV are colored as in gray. The surface representations of CIII
822
and CIV are colored as in cyan, the upstream DNA of Mor-TAC or C-TAC was shown as cartoon
823
in gray or red, respectively; (B) Alignment between the cryo-EM structures of RPo-rpsT (PDB
824
ID: 6OUL, gray) and Mor-TAC(salmon) or C-TAC(firebrick) on the upstream DNA (left panel),
825
the structure of Mor-TAC, C-TAC was superimposed on the structure of RPo-rpsT, The
826
superimpositions were performed using σ70R4 atoms. (C) Proposed transcription activation
827
model for the phage Mu Mor/C family proteins. Compared to a canonical RPo (left panel),
828
central symmetrically arranged Mor homotetramer hijacks E. coli RNAP through interacting
829
with its conserved domains (αCTD and σ70R4) and recruits RNAP to activate transcription of
830
the phage Mu middle promoter through engaging promoter DNA (middle panel). In contrast,
831
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33
central symmetrically arranged C homotetramer makes diverse interactions with the conserved
832
domains (αCTD, σ70R4 and β FTH) of RNAP and significantly bends the upstream promoter
833
DNA, which cooperatively recruit RNAP to activate transcription of the phage Mu late
834
promoter (right panel).
835
836
837
838
839
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