Studies on the expression of 6S RNA from E. coli: involvement of regulators important for stress and growth adaptation.

The small bacterial 6S RNA has been recognized as a transcriptional regulator, facilitating the transition from exponential to stationary growth phase by preferentially inhibiting E sigma 70 RNA polymerase holoenzyme transcription. Consistent with this function, the cellular concentration of 6S RNA increases with stationary phase. We have studied the underlying mechanisms responsible for the growth phase-dependent differences in 6S RNA concentration. To this aim, we have analyzed the effects of the typical bacterial growth phase and stress regulators FIS, H-NS, LRP and StpA on 6S RNA expression. Measurements of 6S RNA accumulation in strains deficient in each one of these proteins support their contribution as potential regulators. Specific binding of the four proteins to DNA fragments containing 6S RNA promoters was demonstrated by gel retardation and DNase I footprinting. Moreover, in vitro transcription analysis with both RNA polymerase holoenzymes, E sigma 70 and E sigma 38, demonstrated a direct inhibition of 6S RNA transcription by H-NS, StpA and LRP, while FIS seems to act as a dual regulator. In vitro transcription in the presence of ppGpp indicates that 6S RNA promoters are not stringently regulated. Our results underline that regulation of 6S RNA transcription depends on a complex network, involving a set of bacterial regulators with general importance in the adaptation to changing growth conditions.

Studies on the function of the riboregulator 6S RNA from E. coli: RNA polymerase binding, inhibition of in vitro transcription and synthesis of RNA-directed de novo transcripts.

Escherichia coli 6S RNA represents a non-coding RNA (ncRNA), which, based on the conserved secondary structure and previous functional studies, had been suggested to interfere with transcription. Selective inhibition of sigma-70 holoenzymes, preferentially at extended -10 promoters, but not stationary-phase-specific transcription was described, suggesting a direct role of 6S RNA in the transition from exponential to stationary phase. To elucidate the underlying mechanism, we have analysed 6S RNA interactions with different forms of RNA polymerase by gel retardation and crosslinking. Preferred binding of 6S RNA to Esigma(70) was confirmed, however weaker binding to Esigma(38) was also observed. The crosslinking analysis revealed direct contact between a central 6S RNA sequence element and the beta/beta' and sigma subunits. Promoter complex formation and in vitro transcription analysis with exponential- and stationary-phase-specific promoters and the corresponding holoenzymes demonstrated that 6S RNA interferes with transcription initiation but does not generally distinguish between exponential- and stationary-phase-specific promoters. Moreover, we show for the first time that 6S RNA acts as a template for the transcription of defined RNA molecules in the absence of DNA. In conclusion, this study reveals new aspects of 6S RNA function.