The circular bacterial genome DNA exists in cells in the form of nucleoids. In the present study, using genetic, molecular and structural biology techniques, we show that nascent single-stranded RNAs are involved in the step-wise folding of nucleoid fibers. In Escherichia coli, RNase A degraded thicker fibers (30 and 80 nm wide) into thinner fibers (10 nm wide), while RNase III and RNase H degraded 80-nm fibers into 30-nm (but not 10-nm) fibers. Similarly in Staphylococcus aureus, RNase A treatment resulted in 10-nm fibers. Treatment with the transcription inhibitor, rifampicin, in the absence of RNase A changed most nucleoid fibers to 10-nm fibers. Proteinase-K treatment of nucleoids exposed DNA. Thus, the smallest structural unit is an RNase A-resistant 10-nm fiber composed of DNA and proteins, and the hierarchical structure of the bacterial chromosome is controlled by transcription itself. In addition, the formation of 80-nm fibers from 30-nm fibers requires double-stranded RNA and RNA-DNA hetero duplex. RNA is evident in the architecture of log-phase uncondensed and stationary-phase condensed nucleoids.