Pulldown of RNA using PrgU-FLAG from Enterococcus faecalis OG1RF pCF10

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the expression of its Type 4 Secretion System (T4SS) genes, controlled by the PQ promoter. The PQ promoter is strictly regulated, partially to limit cell toxicity caused by overproduction of PrgB, a T4SS adhesin. PrgU plays an important role in regulating this toxicity by decreasing PrgB levels. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. We used a combination of in vivo methods to quantify PrgU effects on prgQ transcripts at both single cell and population levels. Comparison of transcript levels in RNA pulldowns using PrgU-FLAG