Transcriptional initiation of a small RNA, not R‐loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae

Summary Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis‐acting sRNA (G4‐sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4‐sRNA forms a stable RNA:DNA hybrid (R‐loop) with its template strand. However, modulating R‐loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R‐loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R‐loops are not required to promote pilin Av. Neisseria gonorrhoeae, the causative agent of gonorrhea, changes one of its surface exposed structures through DNA recombination, in a process known as antigenic variation. A small RNA is required for antigenic variation and we found that though it forms an RNA:DNA hybrid, hybrid stability is not important for antigenic variation. It is the transcription of the small RNA that allows for the formation of an alternate DNA structure called a guanine quadruplex, that drives antigenic variation.