BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF NUSG PARALOG LOAP
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The NusG family of transcription factors is the only universally conserved family of transcription elongation regulators in all three domains of life. NusG proteins exert ubiquitous genetic regulatory effects by reversibly binding RNA-polymerase (RNAP) during transcription elongation and modulate its function. A phylogenetic analysis of the NusG family of proteins identified several distinct subfamilies of NusG paralogs that are widespread amongst bacterial species. These different NusG paralogs are likely to exert regulatory control over distinct subsets of genes. Yet, despite the importance of the genes they regulate, most of the subfamilies of NusG paralogs (e.g., UpxY, TaA, ActX and LoaP) have not been investigated in depth. Additionally, the regulatory mechanisms that these transcription elongation factors employ are likely to differ between one another to allow for specific recruitment to target operons and prevent competition with the housekeeping NusG factor. The LoaP subfamily of NusG proteins is primarily encoded by Actinobacteria, Firmicutes and Spirochaetes. While regulons for the LoaP subfamily have only been identified in a few organisms, the loaP gene is oftentimes found adjacent to long operons encoding for biosynthesis of secondary metabolites suggesting a regulatory relationship with these pathways. In Bacillus velezensis, LoaP promotes transcription antitermination of two long biosynthetic operons which encode for two different polyketide antibiotics: difficidin and macrolactin. Intriguingly, the cis-determinants for LoaP antitermination include a small RNA hairpin (~26 nts) located within the 5’ leader region of target operons. LoaP associates with the RNA hairpin in vitro with nanomolar affinity and high specificity via basic residues that are highly conserved within the C-terminal KOW domain, in contrast to other well-characterized bacterial NusG proteins which do not exhibit RNA-binding activity. These data indicate that LoaP employs a distinct regulatory mechanism to achieve targeted regulation of large biosynthetic operons in bacteria. Furthermore, this discovery expands the repertoire of macromolecular interactions exhibited by bacterial NusG proteins during transcription elongation to include an RNA ligand. Crystallographic studies of LoaP-RNA complex are in progress, and recent results will be discussed.