Binding and release of the 6S transcriptional control RNA

6S RNA is an important noncoding RNA that regulates eubacterial transcription. In Escherichia coli this RNA binds to the σ 70 RNA polymerase holoenzyme and is released by the synthesis of a short product RNA. In order to determine how binding and release are controlled by the 6S RNA sequence, we used in vitro selection to screen a high diversity library containing ∼4 × 10 12 sequences for functional 6S RNA variants. Residues critical for binding were found to be located in a “-35” region upstream of the 6S RNA transcription bubble mimic structure. Mutating these phylogenetically conserved residues invariably led to decreases in binding and removing them abolished binding, implicating these nucleotides in a biologically important interaction with the Eσ 70 complex. Interestingly, mutation of phylogenetically conserved “-10” residues that were also upstream of the site of pRNA synthesis was found to influence 6S RNA release rates in addition to modulating -35 binding. These results indicate how 6S RNA -35 binding to σ 70 RNA polymerase holoenzyme can regulate expression from “strong” and “weak” -35 DNA promoters and suggest that 6S RNA release rates have been fine tuned over evolutionary time so as to correctly regulate cellular levels of transcription.