Riboswitch and small RNAs modulate btuB translation initiation in Escherichia coli and trigger distinct mRNA regulatory mechanisms.

Small RNAs (sRNAs) and riboswitches represent distinct classes of RNA regulators that control gene expression upon sensing metabolic or environmental variations. While sRNAs and riboswitches regulate gene expression by affecting mRNA and protein levels, existing studies have been limited to the characterization of each regulatory system in isolation, suggesting that sRNAs and riboswitches target distinct mRNA populations. We report that the expression of btuB in Escherichia coli, which is regulated by an adenosylcobalamin (AdoCbl) riboswitch, is also controlled by the small RNAs OmrA and, to a lesser extent, OmrB. Strikingly, we find that the riboswitch and sRNAs reduce mRNA levels through distinct pathways. Our data show that while the riboswitch triggers Rho-dependent transcription termination, sRNAs rely on the degradosome to modulate mRNA levels. Importantly, OmrA pairs with the btuB mRNA through its central region, which is not conserved in OmrB, indicating that these two sRNAs may have specific targets in addition to their common regulon. In contrast to canonical sRNA regulation, we find that OmrA repression of btuB is lost using an mRNA binding-deficient Hfq variant. Together, our study demonstrates that riboswitch and sRNAs modulate btuB expression, providing an example of cis- and trans-acting RNA-based regulatory systems maintaining cellular homeostasis.

Identification and characterization of RNA binding sites for (p)ppGpp using RNA-DRaCALA.

Ligand-binding RNAs (RNA aptamers) are widespread in the three domains of life, serving as sensors of metabolites and other small molecules. When aptamers are embedded within RNA transcripts as components of riboswitches, they can regulate gene expression upon binding their ligands. Previous methods for biochemical validation of computationally predicted aptamers are not well-suited for rapid screening of large numbers of RNA aptamers. Therefore, we utilized DRaCALA (Differential Radial Capillary Action of Ligand Assay), a technique designed originally to study protein-ligand interactions, to examine RNA-ligand binding, permitting rapid screening of dozens of RNA aptamer candidates concurrently. Using this method, which we call RNA-DRaCALA, we screened 30 ykkC family subtype 2a RNA aptamers that were computationally predicted to bind (p)ppGpp. Most of the aptamers bound both ppGpp and pppGpp, but some strongly favored only ppGpp or pppGpp, and some bound neither. Expansion of the number of biochemically verified sites allowed construction of more accurate secondary structure models and prediction of key features in the aptamers that distinguish a ppGpp from a pppGpp binding site. To demonstrate that the method works with other ligands, we also used RNA DRaCALA to analyze aptamer binding by thiamine pyrophosphate.

Stem-Loop Structures within mRNA Coding Sequences Activate Translation Initiation and Mediate Control by Small Regulatory RNAs.

Initiation is the rate-limiting step of translation, and in bacteria, mRNA secondary structure has been extensively reported as limiting the efficiency of translation by occluding the ribosome-binding site. In striking contrast with this inhibitory effect, we report here that stem-loop structures located within coding sequences instead activate translation initiation of the Escherichia coli fepA and bamA mRNAs involved in iron acquisition and outer membrane proteins assembly, respectively. Both structures promote ribosome binding in vitro, independently of their nucleotide sequence. Moreover, two small regulatory RNAs, OmrA and OmrB, base pair to and most likely disrupt the fepA stem-loop structure, thereby repressing FepA synthesis. By expanding our understanding of how mRNA cis-acting elements regulate translation, these data challenge the widespread view of mRNA secondary structures as translation inhibitors and show that translation-activating elements embedded in coding sequences can be targeted by small RNAs to inhibit gene expression.

Mechanistic study of base-pairing small regulatory RNAs in bacteria.

In all three kingdoms of life, RNA is not only involved in the expression of genetic information, but also carries out extremely diverse cellular functions. This versatility is essentially due to the fact that RNA molecules can exploit the power of base pairing to allow them to fold into a wide variety of structures through which they can perform diverse roles, but also to selectively target and bind to other nucleic acids. This is true in particular for bacterial small regulatory RNAs that act by imperfect base-pairing with target mRNAs, and thereby control their expression through different mechanisms. Here we outline an overview of in vivo and in vitro approaches that are currently used to gain mechanistic insights into how these sRNAs control gene expression in bacteria.