In Escherichia coli, 6S RNA functions as a modulator of RNA polymerase sigma70-holoenzyme activity, but its biosynthetic pathway remains uncharacterized. In this study, to further understand the regulatory circuit of 6S RNA biosynthesis for the modulation of Esigma70 activity, we have characterized the biogenesis of 6S RNA. We reveal that there are two different precursors, a long and a short molecule, which are transcribed from the distal P2 and proximal P1 promoter, respectively. Transcription from the P2 promoter is both sigma70- and sigmaS-dependent, whereas, in contrast, P1 transcription is sigma70- but not sigmaS-dependent. Both precursors are processed to generate the 5' end of 6S RNA, and while the long precursor is processed exclusively by RNase E, the short precursor is processed by both RNase G and RNase E. Our data indicate that the switching of the utilization of both sigma factors and endoribonucleases in the biogenesis of 6S RNA would play an essential role in modulating its levels in E.coli.