Binding and release of the 6S transcriptional control RNA

Abstract

ABSTRACT6S RNA is an important noncoding RNA that regulates eubacterial transcription. In Escherichia coli this RNA binds to the s 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 3 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 Es 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 s 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.

E. coli 6S RNA release from RNA polymerase requires σ70 ejection by scrunching and is orchestrated by a conserved RNA hairpin

“…Indeed, T1 RNase digestion of the free 6S RNA (S1) under native conditions revealed G136 to be sensitive to T1 RNase (Fig. 1B), consistent with previous secondary-structure models of the 6S RNA (Barrick et al 2005;Shephard et al 2010). After binding and then releasing the 6S RNA from the holoenzyme via pRNA synthesis so as to form the S4 state, G136 became significantly resistant to T1 RNase while the cleavage pattern of G97, G88, G82, G80, and G79, which were not expected to undergo any secondarystructure rearrangement, remained unaffected.…”

“…The unbound 6S RNA (S1 state) and the final released 6S:pRNA complex (the S4 state) are devoid of protein and provide simple evidence that a structural rearrangement occurs during the process of 6S RNA release from E. coli RNA polymerase. Having shown that the À10 region can alter both binding and release rate (Shephard et al 2010), we noticed together with others (Wurm et al 2010;Beckmann et al 2012), that the top strand of the conserved downstream helix is complementary to the conserved À10 region (Fig. 1A).…”

“…(B) Native T1 RNase digestion of 59 This region, would by geometric analogy to the À10 DNA promoter recognition element (Barrick et al 2005), make interactions with s 70 regions 2.3 and 2.4 and potentially region 3 (Murakami et al 2002). Consistent with this picture, mutating the À10 region can either increase or decrease 6S RNA binding and has been shown to modulate 6S RNA release rate (Shephard et al 2010). The conserved downstream region has only a marginal influence on the initial binding of the 6S RNA to Es 70 .…”

“…A phylogenetically conserved ''À35'' region ( Fig. 1A; Barrick et al 2005) that is located upstream of the large open bubble structure has been demonstrated by in vitro selection, as well as truncation experiments, to be essential for 6S RNA binding to E. coli Es 70 (Shephard et al 2010). This site interacts with the C terminus of s 70 as deleting the 4.2 subdomain precludes 6S RNA binding (Klocko and Wassarman 2009).…”

“…Since this region of the s 70 interacts with both the 6S RNA and the À35 DNA promoter element, a mechanism for the 6S RNA to competitively bind to Es 70 is naturally suggested (Wassarman and Storz 2000;Shephard et al 2010). A poorly conserved bulged helix in the 6S RNA spaces this À35 binding element away from a large open bubble that contains a ''À10'' region that is highly conserved in the g-proteobacteria (Barrick et al 2005;Shephard et al 2010). FIGURE 1.…”