|It is presumed that, in living cells, the initiation of transcription occurs using NTPs alone, i.e. “de novo”. Here we establish in bacterial cells that transcription initiation can also occur via use of small ~2- to 4-nt RNA transcripts which we refer to as “nanoRNAs”. We further show that priming of transcription with nanoRNAs can influence gene expression in bacterial cells. Our findings challenge the conventional paradigm that all cellular transcription occurs de novo. In addition, our findings indicate that nanoRNAs represent a distinct class of functional small RNAs that can affect gene expression through direct incorporation into a target RNA transcript rather than through a traditional antisense-based mechanism.