Bacillus velezensis LoaP Promotes Antitermination of Antibiotic Synthesis Operons by Antagonizing NusA

ABSTRACT: 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 such that it bypasses 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, Bacillus velezensis 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 (dfn) antitermination activity in vitro. The dfn 5’ leader region contains a small RNA hairpin essential for antitermination in vivo and in vitro, 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.

INSTRUMENT(S):

ORGANISM(S): Bacillus Velezensis Fzb42 Bacteria

SUBMITTER: Conor Jenkins  

LAB HEAD: Wade Winkler

PROVIDER: PXD063323 | Pride | 2025-10-09

REPOSITORIES: Pride