The synthesis of ribosomal RNA (rRNA) is a tightly regulated central process in all cells. In bacteria efficient expression of all seven rRNA operons relies on the suppression of termination signals (antitermination) and the proper maturation of the synthesized rRNA. These processes depend on N-utilization substance (Nus) factors A, B, E and G, as well as ribosomal protein S4 and inositol monophosphatase SuhB, but their structural basis is only poorly understood. Combining nuclear magnetic resonance spectroscopy and biochemical approaches we show that Escherichia coli SuhB can be integrated into a Nus factor-, and optionally S4-, containing antitermination complex halted at a ribosomal antitermination signal. We further demonstrate that SuhB specifically binds to the acidic repeat 2 (AR2) domain of the multi-domain protein NusA, an interaction that may be involved in antitermination or posttranscriptional processes. Moreover, we show that SuhB interacts with RNA and weakly associates with RNA polymerase (RNAP). We finally present evidence that SuhB, the C-terminal domain of the RNAP α-subunit, and the N-terminal domain of NusG share binding sites on NusA-AR2 and that all three can release autoinhibition of NusA, indicating that NusA-AR2 serves as versatile recruitment platform for various factors in transcription regulation.
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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