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Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient <jats:italic>pilE<\/jats:italic> gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis\u2010acting sRNA (G4\u2010sRNA) are located upstream of the <jats:italic>pilE<\/jats:italic> 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\u2010sRNA forms a stable RNA:DNA hybrid (R\u2010loop) with its template strand. However, modulating R\u2010loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R\u2010loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R\u2010loops are not required to promote pilin Av.<\/jats:p>","DOI":"10.1111\/mmi.14356","type":"journal-article","created":{"date-parts":[[2019,7,24]],"date-time":"2019-07-24T07:48:53Z","timestamp":1563954533000},"page":"1219-1234","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":19,"title":["Transcriptional initiation of a small RNA, not R\u2010loop stability, dictates the frequency of pilin antigenic variation in <i>Neisseria gonorrhoeae<\/i>"],"prefix":"10.1111","volume":"112","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4053-2645","authenticated-orcid":false,"given":"Lauren L.","family":"Prister","sequence":"first","affiliation":[{"name":"Department of Microbiology\u2010Immunology Northwestern University Feinberg School of Medicine 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