4.1) 6S RNA is regulated simultaneously by H-NS, Lrp and Fis
The E. coli 6S RNA (SsrS) was first described by Brownlee in 1971 (Brownlee, 1971). This 184 nt long sRNA has an extended double-stranded structure with a large single-stranded bulge, similar to the DNA structure in an open promoter complex (Barrick et al. , 2005; Trotochaud and Wassarman, 2005). This secondary structure is a common feature of 6S RNAs, and in E. coli it preferentially interacts with the σ70-RNA polymerase (Eσ 70). This complex leads to the titration of RNAP and thus inhibition of the transcription of σ70-dependent promoters (Wassarman and Storz, 2000). Given that Eσ 70 is an exponential phase-specific polymerase, and that cellular levels of 6S RNA increase during the stationary phase (Wassarman and Storz, 2000), it has been suggested that 6S RNA facilitates the shift of global gene expression in E. coli from the exponential to the stationary phase of growth. In addition, in vivo experiments have shown that 6S RNA serves as a template for the synthesis of a short RNA product (pRNA) that forms the 6S RNA:pRNA during exponential growth, realising the 6S RNA from the RNAP (Bonar et al. , 2022).
The function of 6S RNA depends on its maturation, which involves the sequential removal of the extra 3′ and 5′ sequences by exonucleases and RNase E/G (Kim and Lee, 2004; Chae et al. , 2011). Its transcriptional initiation is under the control of two tandem promoters, P1 and P2, which are regulated by H-NS, Lrp and Fis (Figure 2 ) (Neusser et al. , 2008). The extended region of upstream DNA between the P1 and P2 promoters is occupied by H-NS, resulting in repression of ssrS transcription. Six to seven Lrp binding sites were identified clustered around the two promoters. In vivo andvitro assays confirmed the repression of ssrS transcription initiation by H-NS and Lrp. The Fis protein acts more like a dual regulator activating the P1 promoter but inhibiting the P2 promoter. Fis binding sites are mainly clustered upstream of P1 (Hirvonen et al. , 2001; Hillebrand et al. , 2005), and overlapping and downstream of the P2 promoter (Neusser et al. , 2008). In addition, three sites overlap with Lrp binding sites, suggesting that Fis and Lrp interfere with each other (Neusser et al. , 2008). Although the precise mechanism involved has not been elucidated, it has been shown that transcription from the P1 and P2 promoters is feedback-activated and feedback-inhibited depending on the cellular context. This feedback regulation disappears in Δfis strains, suggesting that Fis is involved in the regulatory circuitry that ensures an appropriate cellular concentration of 6S RNA (Leeet al. , 2013). These findings underline that the regulation of 6S RNA transcription is under the control of a sophisticated network of bacterial regulation, which plays a pivotal role in facilitating adaptation to fluctuating growth conditions.