Bacterial RNA motif in the 5′ UTR of rpsF interacts with an S6:S18 complex

Abstract

Approximately half the transcripts encoding ribosomal proteins in Escherichia coli include a structured RNA motif that interacts with a specific ribosomal protein to inhibit gene expression, thus allowing stoichiometric production of ribosome components. However, many of these RNA structures are not widely distributed across bacterial phyla. It is increasingly common for RNA motifs associated with ribosomal protein genes to be identified using comparative genomic methods, yet these are rarely experimentally validated. In this work, we characterize one such motif that precedes operons containing rpsF and rpsR, which encode ribosomal proteins S6 and S18. This RNA structure is widely distributed across many phyla of bacteria despite differences within the downstream operon, and examples are present in both E. coli and Bacillus subtilis. We demonstrate a direct interaction between an example of the RNA from B. subtilis and an S6:S18 complex using in vitro binding assays, verify our predicted secondary structure, and identify a putative protein-binding site. The proposed binding site bears a strong resemblance to the S18 binding site within the 16S rRNA, suggesting molecular mimicry. This interaction is a valuable addition to the canon of ribosomal protein mRNA interactions. This work shows how experimental verification translates computational results into concrete knowledge of biological systems.

Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria

“…Whether every operon encoding r-protein(s) is regulated by feedback inhibition or, in some cases, the adequate level of r-protein synthesis could be achieved by other means (e.g., rapid turnover of proteins) cannot be predicted with a certainty, and each case should be experimentally validated. In this regard, our results on the rplY in vivo regulation (this paper) together with the recent in vitro data suggesting the S6:S18-mediated control of the rpsF-priBrpsR-rplI operon (Matelska et al 2013;Fu et al 2014) expand our knowledge of the regulatory circuits controlling ribosome biogenesis in bacteria.…”

“…This regulatory strategy has been previously demonstrated for many r-protein operons from E. coli and, in some cases, from other bacterial species (Zengel and Lindahl 1994;Allen et al 1999Allen et al , 2004Boni et al 2001;Tchufistova et al 2003;Guillier et al 2005;Aseev et al 2008Aseev et al , 2009Fu et al 2013Fu et al , 2014. However, the overall picture remains incomplete, as information is available only for a half of E. coli r-protein operons.…”

“…Regulation of r-protein operons is provided by specific cis-regulatory RNA elements which, as a rule, are more or less widely conserved in γ-proteobacteria (Fu et al 2013(Fu et al , 2014. Phylogenetic analysis of the rplY mRNA 5 ′ UTR revealed conservation of several regulatory RNA motifs including an atypical SD-sequence in a subset of γ-proteobacterial families, viz.…”

“…This hairpin seems to be required only for the L25-mediated control but not for translation efficiency, since its deletions increased the translation yield, simultaneously decreasing autogenous repression. At first glance, a set of base parings within HII does not resemble the L25 target on 5S rRNA, the loop E, so that at present it is hard to suggest (or to rule out) that binding of L25 to its mRNA mimics its interaction with 5S rRNA, following the principle of "molecular mimicry" earlier proposed for a number of r-protein operons (Nomura et al 1980;Stelzl et al 2003;Mathy et al 2004;Guillier et al 2005;Fu et al 2014).…”

An S6:S18 complex inhibits translation of E. coli rpsF

“…We and others recently reported a conserved RNA structure preceding rpsF, which encodes ribosomal protein S6 (Matelska et al 2013;Fu et al 2014). The RNA structure (rpsF_leader) is widely distributed to many bacterial species and displays some similarity with the S6:S18-binding site on the 16S rRNA.…”