Structural basis for λN-dependent processive transcription antitermination

Said, Nelly; Krupp, Ferdinand; Anedchenko, E. A.; Santos, K.F.; Dybkov, Olexandr; Huang, Yong-Heng; Lee, Chung-Tien; Loll, Bernhard; Behrmann, Elmar; Bürger, Jörg; Mielke, Thorsten; Loerke, Justus; Urlaub, Henning; Spahn, C.M.T.; Weber, Gert; Wahl, Markus C.

doi:10.1038/nmicrobiol.2017.62

Cited by 59 publications

(102 citation statements)

References 89 publications

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“…Protein N from phage λ was the first antiterminator protein to be discovered 22 , which together with Nus factors A, B, C, and G, and RNAP forms a transcription antitermination complex (TAC) 28 . The structural basis for this form of antitermination was recently described 163 . As the elongating TAC encounters an intrinsic terminator, the S1 domain of NusA prevents terminator formation by sequestering the upstream arm of the terminator hairpin.…”

Section: Text Box 1: Rbps Expressed From Plasmids and Phagesmentioning

confidence: 99%

RNA-binding proteins in bacteria

2018

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RNA-binding proteins (RBPs) are central to most if not all cellular processes, dictating the fate of virtually all RNA molecules in the cell. Starting with pioneering work on ribosomal proteins, studies of bacterial RBPs have paved the way for molecular studies of RNA-protein interactions. Work over the years has identified major RBPs that act on cellular transcripts at the various stages of bacterial gene expression and that enable their integration into post-transcriptional networks that also comprise small non-coding RNAs. Bacterial RBP research has now entered a new era in which RNA sequencing-based methods permit mapping of RBP activity in a truly global manner in vivo. Moreover, the soaring interest in understudied members of host-associated microbiota and environmental communities is likely to unveil new RBPs and to greatly expand our knowledge of RNA-protein interactions in bacteria.

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Section: Text Box 1: Rbps Expressed From Plasmids and Phagesmentioning

confidence: 99%

RNA-binding proteins in bacteria

2018

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“…4A) based on a recent cryo-EM structure of the E. coli TEC (17) using our ops9:RfaH structure. Since NusG and its homologs share the RNAP-binding mode (6,16,25,26), the crystal structure of Pyrococcus furiosus Spt5 bound to the RNAP clamp domain (3,27) served as a template for modeling. The NT DNA hairpin observed in the ops9:RfaH structure could be readily modeled into the TEC.…”

Section: Cd)mentioning

confidence: 99%

“…The NTD binds across the DNA-binding channel, bridging the RNAP pincers composed of the β' clamp and β lobe domains and locking elongating RNAP in a pause-resistant state (2), a mechanism likened to that of processivity clamps in DNA polymerases (3). The CTDs modulate RNA synthesis by making contacts to nucleic acids or to proteins involved in diverse cellular processes; Escherichia coli NusG binds either to termination factor Rho to silence aberrant transcription (4,5) or to ribosomal protein S10 to promote antitermination (6) and transcription-translation coupling (7).…”

Section: Introductionmentioning

confidence: 99%

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The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

Zuber

Schweimer

NandyMazumdar

et al. 2018

Preprint

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21RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique 22 mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function 23 depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template 24 DNA strand of the transcription bubble. We used structural and functional analyses to elucidate 25 the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate 26 RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms 27 a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is 28 specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion 29 that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the 30 structure of the non-template strand are read out by transcription factors, expanding the 31 repertoire of transcriptional regulators in all domains of life. 32 33 72 (Kohler et al., 2017; Saxena et al., 2018). Following TEC dissociation, RfaH has been proposed 73 to regain the autoinhibited state (Tomar et al., 2013), thus completing the cycle. 74 A model of E. coli RfaH bound to Thermus thermophilus TEC was constructed by arbitrarily 75 threading the NT DNA (absent in the X-ray structure) through the TEC (Belogurov et al., 2007). 76 While subsequent functional analysis of RfaH supports this model (Belogurov et al., 2010), the 77 path of the NT DNA and the details of ops:RfaH interactions remain unclear. The NT DNA is 78 flexible in the TEC (Kang et al., 2017) and could be trapped in a state incompatible with 79 5 productive elongation; RfaH/NusG and yeast Spt5 have been proposed to constrain the NT 80 strand to increase processivity (Crickard et al., 2016; NandyMazumdar et al., 2016). Direct 81 contacts to the NT DNA have been demonstrated recently for Bacillus subtilis NusG (Yakhnin et 82 al., 2016) and Saccharomyces cerevisiae Spt5 (Crickard et al., 2016). 83 Here we combined structural and functional analyses to dissect RfaH:ops interactions. Our data 84 argue that ops plays two roles in RfaH recruitment: it halts RNAP to aid loading of RfaH and 85 makes specific contacts with RfaH-NTD. Strikingly, we found that a small hairpin extruded from 86 the NT DNA is required for RfaH recruitment, demonstrating how NT DNA flexibility could be 87 harnessed for transcriptional regulation in this and potentially many other systems. 88 127 pausing (Figure 2D and Figure 2 -figure supplement 1), consistent with their variability in the 128 consensus pause sequence (Figure 2A). Conversely, the G12C substitution eliminated the pause 129 at U11, making measurements of RfaH antipausing activity unreliable, but did not abrogate RfaH 130 recruitment (Figures 2C,D), suggesting that pausing at U11 is dispensable for RfaH binding 131 when RNAP is transcribing slowly. 132 Observations that RfaH is recruited to RNAP transcribing the G12C template raised a possibi...

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“…NusE is the active component in processive AT whereas NusB supports loading of NusE to the transcription machinery 27 . NusG-CTD can interact with NusE and thus anchors the NusE:NusB:boxA complex to the RNAP 6,7,24 . NusG-mediated tethering of NusE:NusB:boxA to the RNAP might also be important for ribosomal AT 17,18 .…”

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confidence: 99%

NusA directly interacts with antitermination factor Q from phage λ

Dudenhoeffer

Borggraefe

Schweimer

et al. 2020

Sci Rep

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Antitermination (At) is a ubiquitous principle in the regulation of bacterial transcription to suppress termination signals. in phage λ antiterminator protein Q controls the expression of the phage's late genes with loading of λQ onto the transcription elongation complex halted at a σ-dependent pause requiring a specific DNA element. The molecular basis of λQ-dependent At and its dependence on n-utilization substance (nus) A is so far only poorly understood. Here we used solution-state nuclear magnetic resonance spectroscopy to show that the solution structure of λQ is in agreement with the crystal structure of an N-terminally truncated variant and that the 60 residues at the N-terminus are unstructured. We also provide evidence that multidomain protein nusA interacts directly with λQ via its N-terminal domain (NTD) and the acidic repeat (AR) 2 domain, with the λQ:NusA-AR2 interaction being able to release NusA autoinhibition. The binding sites for NusA-NTD and NusA-AR2 on λQ overlap and the interactions are mutually exclusive with similar affinities, suggesting distinct roles during λQ-dependent At, e.g. the λQ:nusA-ntD interaction might position nusA-ntD in a way to suppress termination, making nusA-ntD repositioning a general scheme in At mechanisms. Transcription of all cellular genomes is mediated by evolutionary related multisubunit RNA polymerases (RNAPs) 1. RNA synthesis is a discontinuous process that underlies tight regulation by various transcription factors that bind to RNAP, affecting its processivity. In Gram-negative bacteria the core RNAP consists of five subunits (2 x α, β, β' , ω). The flap region of the β subunit forms the outer wall of the RNA exit channel with the β flap tip helix (βFTH) at the top of this region being able to regulate the width of the channel, making it a key regulatory element 2-8. Antitermination (AT) is a ubiquitous mechanism to suppress termination signals and is widely used in bacteria. AT has first been described for bacteriophage λ where it controls the expression of early and late genes, being thus essential for the life cycle of the phage 9. Phage λ uses two AT mechanisms which involve either antiterminator protein N or antiterminator protein Q. In λN-dependent AT the intrinsically disordered protein N is recruited to elongating RNAP by an AT signal in the nascent RNA and forms a complex with RNAP and the Escherichia coli (E. coli) host factors N-utilization substances (Nus) A, B, E, and G 6,7. In this transcription AT complex (TAC) λN repositions NusA and remodels the βFTH, enabling the TAC to read through termination signals by preventing the formation of pause/terminator hairpins 6,7. In λQ-dependent AT protein λQ requires a λQ binding element (QBE) on the DNA for recruitment and is loaded onto RNAP halted at an adjacent sigma-dependent promoter-proximal pause site 10,11. Recent cryo electron microscopy (EM) studies of the AT mechanism of protein Q from phage 21, Q21, revealed that two Q21 proteins engage with RNAP in a Q21-TAC, one of which forms a torus at the ...

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Structural basis for λN-dependent processive transcription antitermination

Cited by 59 publications

References 89 publications

RNA-binding proteins in bacteria

RNA-binding proteins in bacteria

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand

NusA directly interacts with antitermination factor Q from phage λ

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