NusA directly interacts with antitermination factor Q from phage λ

Abstract

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 ...

Transcription complexes as RNA chaperones

“…It will be interesting to see in the future whether 82Q-like proteins act by mechanisms distinct from those observed for 21Q. Furthermore, it has been shown that λQ directly interacts with NusA [ 110 ], and it remains to be seen whether and how this interaction on a λQ-EC modulates NusA’s co-transcriptional RNA chaperoning activity.…”