<i>Bacillus velezensis</i> LoaP promotes antitermination by antagonizing NusA

The transcription factor NusG is essential for the bacterial transcription elongation complex (TEC). During elongation, NusG dynamically interacts with other proteins, affecting transcription-translation coupling, processivity, and termination. NusG is integral to antitermination complexes, where it associates with other factors to modify the TEC, allowing it to bypass termination sites. In Gammaproteobacteria, these complexes enhance the expression of ribosomal RNA operons, bacteriophage operons, and CRISPR arrays. Specialized NusG paralogs, such as LoaP, RfaH, and UpxY, are believed to form their own unique antitermination complexes. RfaH, the only well-studied paralog, binds to a paused TEC via a specific nontemplate DNA sequence. The RfaH-bound TEC then enhances transcription-translation coupling while also preventing Rho termination. However, it is unclear whether the other NusG paralogs use similar mechanisms, nor have antitermination complexes been investigated for Gram-positive bacteria. Previously, <i>Bacillus velezensis</i> LoaP was shown to enhance transcription of the difficidin antibiotic synthesis operon, but its antitermination determinants were unknown. In this study, we purified RNA polymerase and transcription factors and reconstituted difficidin operon (<i>dfn</i>) antitermination activity <i>in vitro</i>. The <i>dfn</i> 5' leader region contains a small RNA hairpin essential for antitermination <i>in vivo</i> and <i>in vitro</i>, and an intrinsic terminator that is highly dependent on NusA. The antitermination activity of LoaP emanates from its ability to specifically antagonize NusA; no other factors are required. 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 paradigm.IMPORTANCEGenes encoding the NusG paralog LoaP are widely found in Bacillota, Actinomycetota, and Spirochaetota, where they are located near biosynthetic operons for secondary metabolites. Previously, <i>Bacillus velezensis</i> LoaP was shown to promote readthrough of transcription termination sites located within operons encoding for the biosynthesis of the antibiotics difficidin and macrolactin. Thus, some secondary metabolites are produced from terminator-laden operons that rely on antitermination factors such as LoaP. In this study, we show that LoaP bypasses termination by specifically antagonizing the transcription factor NusA. This demonstrates that the LoaP antitermination complex is simpler than expected and that additional factors are not required. Together, our data expand the repertoire of NusG specialized paralogs and the antitermination mechanisms they utilize. Understanding this molecular mechanism will aid in the discovery of natural products regulated by LoaP proteins and assist in the development of new synthetic biology tools that improve the heterologous expression of natural products.