Small, but in control

Small (100–500 nucleotides) regulatory non-coding RNAs (sRNA) are present in bacteria, eukaryotes and archaea. The functions of sRNAs were unknown 35 years ago, when the 6S RNA from Escherichia coli was sequenced. Since then, bacterial sRNAs have been shown to regulate processes such as the transition from growth to stationary phase, quorum sensing and virulence.

6S RNA targets the bacterial RNA polymerase (RNAP). RNAP is composed of a core enzyme (E) and a specificity subunit (σ), which form an Eσ complex that initiates transcription. σ⁷⁰ is the primary factor used for initiating transcription under all growth conditions. 6S RNA forms a stable complex with Eσ⁷⁰, which results in the direct inhibition of transcription during the bacterial stationary phase. By contrast, 6S RNA indirectly activates transcription at promoters requiring a different σ complex, Eσ^S, that is important for survival during the stationary phase. Therefore, 6S RNA regulation of transcription occurs at multiple levels and is required for long-term persistence and survival of E. coli when nutrients are scarce.

To define the conserved properties of 6S RNA, the Wassarman group, reporting in Nature Structural & Molecular Biology, and the Breaker laboratory, reporting in RNA, carried out a phylogenetic analysis of 6S RNAs from many bacterial species. They identified a conserved secondary structure containing a double-stranded region and a central, single-stranded bulge.

To determine which features were essential for regulating transcription, the Wassarman group used in vivo and in vitro studies of mutant 6S RNAs. They showed that reducing the size of the single-stranded region or generating base pairs within the bulge destroyed the ability of 6S RNA to bind to RNAP and to inhibit transcription. By contrast, increasing the size of the bulge or introducing mutations within this region that change the sequence but retain its single-stranded nature did not affect 6S RNA function. The authors also showed that sequence and/or structural information present in the duplex region surrounding the bulge enhanced the activity of the 6S RNA. These data indicate that the structure of the RNA is essential for its activity.

In reconstitution assays, E. coli 6S RNA interacted exclusively with Eσ⁷⁰ but not with Eσ^S or free σ⁷⁰. Furthermore, the formation of the 6S–Eσ⁷⁰ complex in vitro led to the inhibition of transcription. The RNA adopts a structure reminiscent of that of an open promoter complex found during transcription initiation. This similarity of structure led to a proposed model that transcriptional inhibition occurs by direct competition between the promoter DNA and 6S RNA for Eσ⁷⁰. Further experiments are needed to verify this hypothesis.

In addition to two 6S RNAs, another RNA was identified in Bacillus subtilis that had altered RNAP-binding specificity. How this RNA functions is not known, but its existence indicates that there are multiple levels of transcriptional regulation by non-coding RNAs. 6S RNA, in addition to the eukaryotic 7SK and B2 RNAs, make up the short, but growing, list of non-coding RNAs that control gene expression by interacting directly with the transcription machinery.