Abstract
6S RNA is an important noncoding RNA that regulates eubacterial transcription. In Escherichia coli this RNA binds to the sigma(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 approximately 4 x 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 Esigma(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 sigma(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.