NusA directly interacts with antitermination factor Q from phage \u03bb

Benjamin R Dudenhoeffer,Jan Borggraefe,Stefan H Knauer,Kristian Schweimer

doi:10.1038/s41598-020-63523-5

Benjamin R Dudenhoeffer, Jan Borggraefe + Show 2 more

Open Access

https://doi.org/10.1038/s41598-020-63523-5

Copy DOI

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.

Highlights

Transcription of all cellular genomes is mediated by evolutionary related multisubunit RNA polymerases (RNAPs)[1]
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-transcription AT complex (TAC), one of which forms a torus at the RNA exit channel, narrowing it to prevent the formation of pause/terminator hairpins[12,13]
We show that binding of λQ to NusA-AR2 releases autoinhibition of NusA, implying that NusA-AR2 is a versatile interaction platform for various transcription partners, but suggesting that NusA may be regarded as regulatory subunit of RNAP that substitutes the sigma factor

Summary

Introduction

Transcription of all cellular genomes is mediated by evolutionary related multisubunit RNA polymerases (RNAPs)[1]. 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 G6,7 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 ribosomal AT, for example, RNAP pauses at an AT signal and a TAC is formed that contains Nus factors A, B, E, and G as well as further components such as ribosomal protein S4 and inositol monophosphatase SuhB17–20 If this TAC is only responsible for AT or involved in posttranscriptional activities, e.g. RNA maturation, is yet unclear

Methods

Results

Conclusion