Communication between RNA folding domains revealed by folding of circularly permuted ribozymes.

To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 degrees C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 degrees C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T(1) digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.

Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation.

We used a high flux synchrotron X-ray beam to map the structure of 16S rRNA and RNase P in viable bacteria in situ. A 300 ms exposure to the X-ray beam was sufficient for optimal cleavage of the phosphodiester backbone. The in vivo footprints of the 16S rRNA in frozen cells were similar to those obtained in vitro and were consistent with the predicted accessibility of the RNA backbone to hydroxyl radical. Protection or enhanced cleavage of certain nucleotides in vivo can be explained by interactions with tRNA and perturbation of the subunit interface. Thus, short exposures to a synchrotron X-ray beam can footprint the tertiary structure and protein contacts of RNA-protein complexes with nucleotide resolution in living cells.

Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation.

The effectiveness and economics of polyvinyl sulfonic acid (PVSA) as a ribonuclease inhibitor for in vitro systems is reported. PVSA was shown to inhibit RNA cleavage in the presence of RNase A as well as in the presence of Escherichia coli lysate, suggesting that PVSA can act as a broader ribonuclease inhibitor. In addition, PVSA was shown to improve the integrity of mRNA transcripts by up to 5-fold in vitro as measured by their translational viability. Improved preservation of mRNA transcripts in the presence of PVSA under common RNA storage conditions is also reported. A cost comparison with commercially available RNAse inhibitors indicates the economic practicality of PVSA which is approximately 1,700 times less expensive than commonly used ribonuclease inhibitors. PVSA can also be separated from RNA by alcohol precipitation for applications that may be sensitive to the presence of PVSA.

A Modular Genetic System for High-Throughput Profiling and Engineering of Multi-Target Small RNAs.

RNA biology and RNA engineering are subjects of growing interest due to recent advances in our understanding of the diverse cellular functions of RNAs, including their roles as genetic regulators. The noncoding small RNAs (sRNAs) of bacteria are a fundamental basis of regulatory control that can regulate gene expression via antisense base-pairing to one or more target mRNAs. The sRNAs can be customized to generate a range of mRNA translation rates and stabilities. The sRNAs can be applied as a platform for metabolic engineering, to control expression of genes of interest by following relatively straightforward design rules (Kushwaha et al., ACS Synth Biol 5:795-809, 2016). However, the ab initio design of functional sRNAs to precise specifications of gene control is not yet possible. Consequently, there is a need for tools to rapidly profile uncharacterized sRNAs in vivo, to screen sRNAs against "new/novel" targets, and (in the case of metabolic engineering) to develop engineered sRNAs for regulatory function against multiple desired mRNA targets. To address this unmet need, we previously constructed a modular genetic system for assaying sRNA activity in vivo against specifiable mRNA sequences, using microtiter plate assays for high-throughput productivity. This sRNA design platform consists of three modular plasmids: one plasmid contains an inducible sRNA and the RNA chaperone Hfq; the second contains an inducible fluorescent reporter protein and a LacY mutant transporter protein for inducer molecules; and the third plasmid contains a second inducible fluorescent reporter protein. The second reporter gene makes it possible to screen for sRNA regulators that have activity against multiple mRNAs. We describe the protocol for engineering sRNAs with novel regulatory activity using this system. This sRNA prototyping regimen could also be employed for validating predicted mRNA targets of uncharacterized, naturally occurring sRNAs or for testing hypotheses about the predicted roles of genes, including essential genes, in cellular metabolism and other processes, by using customized antisense sRNAs to knock down or tune down gene expression.

High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites.

Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.

Retargeting a Dual-Acting sRNA for Multiple mRNA Transcript Regulation.

Multitargeting small regulatory RNAs (sRNAs) represent a potentially useful tool for metabolic engineering applications. Natural multitargeting sRNAs govern bacterial gene expression by binding to the translation initiation regions of protein-coding mRNAs through base pairing. We designed an Escherichia coli based genetic system to create and assay dual-acting retargeted-sRNA variants. The variants can be assayed for coordinate translational regulation of two alternate mRNA leaders fused to independent reporter genes. Accordingly, we began with the well-characterized E. coli native DsrA sRNA. The merits of using DsrA include its well-characterized separation of function into two independently folded stem-loop domains, wherein alterations at one stem do not necessarily abolish activity at the other stem. Expression of the sRNA and each reporter mRNA was independently controlled by small inducer molecules, allowing precise quantification of the regulatory effects of each sRNA:mRNA interaction in vivo with a microtiter plate assay. Using this system, we semirationally designed DsrA variants screened in E. coli for their ability to regulate key mRNA leader sequences from the Clostridium acetobutylicum n-butanol synthesis pathway. To coordinate intervention at two points in a metabolic pathway, we created bifunctional sRNA prototypes by combining sequences from two singly retargeted DsrA variants. This approach constitutes a platform for designing sRNAs to specifically target arbitrary mRNA transcript sequences, and thus provides a generalizable tool for retargeting and characterizing multitarget sRNAs for metabolic engineering.

Triple-acting Lytic Enzyme Treatment of Drug-Resistant and Intracellular Staphylococcus aureus.

Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA.

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.

Twins, quadruplexes, and more: functional aspects of native and engineered RNA self-assembly in vivo.

The primacy and power of RNA in governing many processes of life has begun to be more fully appreciated in both the discovery and inventive sciences. A variety of RNA interactions regulate gene expression, and structural self-assembly underlies many of these processes. The understanding sparked by these discoveries has inspired and informed the engineering of novel RNA structures, control elements, and genetic circuits in cells. Many of these engineered systems are built up fundamentally from RNA-RNA interactions, often combining modular, rational design with functional selection and screening. It is therefore useful to review the particular class of RNA-based regulatory mechanisms that rely on RNA self-assembly either through homomeric (self-self) or heteromeric (self-nonself) RNA-RNA interactions. Structures and sequence elements within individual RNAs create a basis for the pairing interactions, and in some instances can even lead to the formation of RNA polymers. Example systems of dimers, multimers, and polymers are reviewed in this article in the context of natural systems, wherein the function and impact of self-assemblies are understood. Following this, a brief overview is presented of specific engineered RNA self-assembly systems implemented in vivo, with lessons learned from both discovery and engineering approaches to RNA-RNA self-assembly.

The small noncoding DsrA RNA is an acid resistance regulator in Escherichia coli.

DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (sigmas), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.