cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

This work was supported by the European Union Seventh Framework Programme (FP7/2007–2013), through the European Research Council (232913); Fundación Botín, the Spanish Ministry of Economy and Competitiveness (BIO2007-61762); National Plan of R + D + i; ISCIII—Subdirección General de Evaluación y Fomento de la Investigación (PI10/01702); European Regional Development Fund (to the Institució Catalana de Recerca i Estudis Avançats research professor L.S.); and Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017” (SEV-2012-0208). A.L. received grant BFU2012-39816-C02-01 from the Spanish Ministry of Economy and Competitivity cofinanced by FEDER (Fondo Europeo de Desarrollo Regional) funds.