RNA-binding proteins play vital roles in regulating gene expression and cellular physiology in all organisms. Bacterial RNA-binding proteins can regulate transcription termination via attenuation or antitermination mechanisms, while others can repress or activate translation initiation by affecting ribosome binding. The RNA targets for these proteins include short repeated sequences, longer single-stranded sequences, RNA secondary or tertiary structure, and a combination of these features. The activity of these proteins can be influenced by binding of metabolites, small RNAs, or other proteins, as well as by phosphorylation events. Some of these proteins regulate specific genes, while others function as global regulators. As the regulatory mechanisms, components, targets, and signaling circuitry surrounding RNA-binding proteins have become better understood, in part through rapid advances provided by systems approaches, a sense of the true nature of biological complexity is becoming apparent, which we attempt to capture for the reader of this review.

Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli. While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pK a s in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli , a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E. coli RapA–RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM. These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. Our results along with DNA-binding assays reveal that although RapA binds RNAP away from the DNA-binding main channel, its binding can allosterically close the RNAP clamp, thereby preventing its nonspecific DNA binding and PTC formation. Taken together, we propose that RapA acts as a guardian of RNAP by which RapA prevents nonspecific DNA binding of RNAP without affecting the binding of promoter DNA recognition σ factor, thereby enhancing RNAP recycling.

The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In Escherichia coli, CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified ymdA as having the strongest CsrA-mediated activation across the E. coli transcriptome. Here, we determined that CsrA activates ymdA expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and in vitro coupled transcription-translation assays identified two CsrA binding sites in the leader region of the ymdA transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates ymdA expression at the posttranscriptional level in vivo. Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of ymdA mRNA. RNA structure prediction revealed an RNA hairpin upstream of the ymdA start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of ymdA expression may repress biofilm formation under certain conditions. IMPORTANCE The Csr system of E. coli controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5′ untranslated leader of ymdA mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.

After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined Post-Termination Complex (PTC), limiting free RNAP pool and making transcription inefficient. In Escherichia coli, a Swi2/Snf2 ATPase RapA is involved in countering such inefficiency through RNAP recycling. To understand its mechanism of RNAP recycling, we have determined the cryo-electron microscopy (cryo-EM) structures of two sets of E. coli RapA-RNAP complexes along with RNAP core enzyme and elongation complex (EC). The structures revealed the large conformational changes of RNAP and RapA upon their association implicated in the hindrance in PTC formation. Our study reveals that although RapA binds away from the DNA binding channel of RNAP, it can close the RNAP clamp allosterically thereby preventing its non-specific DNA binding. Together with DNA binding assays, we propose that RapA acts as a guardian of RNAP by which prevents non-specific DNA binding of RNAP without affecting the sigma factor binding to RNAP core enzyme, thereby enhancing RNAP recycling.