INVESTIGATION OF THE NOVEL ANTITERMINATION MECHANISM OF LOAP, A NUSG SPECIALIZED PARALOG

Abstract

The transcription factor NusG plays an important role in the transcription elongation complex across all domains of life. During elongation, the NusG-NTD forms contacts to RNAP while the NusG-CTD dynamically interacts with other proteins, affecting processivity, transcription termination, and in some bacteria, transcription-translation coupling. NusG is integral to antitermination complexes, where it associates with additional factors to modify the TEC in order to bypass termination events at distinct gene clusters. In Gammaproteobacteria, these complexes enhance the expression of ribosomal RNA operons, bacteriophage operons, and CRISPR arrays. Specialized NusG paralogs, such as LoaP, RfaH, or UpxY, are widespread in bacteria and likely form their own unique antitermination complexes. RfaH, the only well-studied paralog, binds to a paused TEC via a specific non-template DNA sequence, then prevents Rho termination while enhancing transcription-translation coupling of targeted operons. It is unclear whether other NusG paralogs use similar mechanisms. LoaP is primarily encoded by Bacillota, Actinomycetota and Spirochaetota, and while regulons influenced by LoaP have only been identified in a few organisms, the recurring location of loaP adjacent to long operons responsible for the biosynthesis of secondary metabolites suggests a regulatory relationship. Previously, Bacillus velezensis LoaP was shown to enhance transcription of the difficidin and macrolactin antibiotic synthesis operons, but its antitermination determinants were unknown. Presented herein is the reconstitution of difficidin operon (dfn) antitermination activity in vitro following the purification of RNA polymerase and additional transcription factors encoded by Bacilli. The dfn 5’ leader region contains a small RNA hairpin ligand essential for LoaP antitermination activity both in vivo and in vitro, and an intrinsic terminator that is highly dependent on NusA. The addition of LoaP specifically antagonizes NusA, thereby promoting readthrough of the termination region without the need for additional factors. These findings demonstrate the basic requirements of the LoaP class of NusG paralogs and indicate that the LoaP antitermination mechanism differs significantly from that of the RfaH – and more broadly, the Gram-positive – paradigm.