Beatrix Suess

Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.

Engineered riboswitches: Expanding researchers’ toolbox with synthetic RNA regulators

Riboswitches are natural RNA‐based genetic switches that sense small‐molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within the last years, several engineered riboswitches have been developed that act on various stages of gene expression. These switches can be engineered to respond to any ligand of choice and are therefore of great interest for synthetic biology. In this review, we present an overview of engineered riboswitches and discuss their application in conditional gene expression systems. We will provide structural and mechanistic insights and point out problems and recent trends in the development of engineered riboswitches.

Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast

Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression.

Engineered riboswitches: overview, problems and trends.

The first conditional gene expression system which employed a small molecule binding aptamer was developed several years before the discovery of natural riboswitches. With the discovery of riboswitches it became obvious that nature uses exactly the same principal of direct RNA-ligand interaction to regulate gene expression in a highly efficient, precise and fast way. In the last decade, further engineered riboswitches have been developed to control gene expression in different organisms. The successful development of new engineered riboswitches, however, is not only dependent on an innovative design but also necessitates a two step process: first, an in vitro selection which results in aptamers with high affinity binding to a desired ligand and second, a subsequent screen to identify RNAs with a desired functionality within cells. This review will give an overview of recent reports of engineered riboswitches, highlight recent developments and point out trends and problems in the field.

Tetracycline-aptamer-mediated translational regulation in yeast.

We describe post‐transcriptional gene regulation in yeast based on direct RNA–ligand interaction. Tetracycline‐dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5′ untranslated region (5′UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap‐distal) in the 5′UTR was more effective than repression via a cap‐proximal position. On the other hand, suppression in a cell‐free system reached maximally 50‐fold and was most effective via a cap‐proximal aptamer. Examination of the kinetics of tetracycline‐dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell‐extracts. Tetracycline‐dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap‐proximal insertion inhibits binding of the 43S complex to the cap structure whereas start‐codon‐proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.

Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch.

Riboswitches are highly structured RNA motifs with gene regulatory activity located in the untranslated regions of mRNAs. They either modulate transcription termination or translation initiation through conformational changes triggered by direct interactions with small metabolite ligands. Many naturally occurring riboswitches are large and structurally very complex. In contrast, synthetic riboswitches—tailored gene regulatory elements for synthetic biology applications—are based on small in vitro selected RNA aptamers. Yet, despite a ligand affinity and specificity comparable to their natural counterparts only a few in vitro selected aptamers are regulatory active in vivo. Recently, Suess et al. engineered a riboswitch for the aminoglycoside antibiotic neomycin B by subjecting an in vitro SELEX-pool to an in vivo screening for gene regulatory activity in a yeastbased reporter gene assay. The resulting neomycin B and ribostamycin (Figure 1a) responsive RNA-element (N1) contains only 27 nucleotides in a bulged hairpin secondary structure (Figure 1b)—the smallest riboswitch functional in vivo identified to date. In sequence and secondary structure, N1 differs completely from an in vitro selected but regulatory inactive RNA-aptamer for the same ligand (R23). Instead it partially resembles the ribosomal A-site, the natural target for aminoglycoside antibiotics (Figure 1b). The NMR spectroscopic analysis of the N1 riboswitch complexed with ribostamycin identifies structural determinants for its regulatory activity and suggests a ligand binding mechanism based on conformational capture. Our results provide insights into the modularity of ligand binding sites in RNA and highlight structural and dynamic features N1 shares with the larger naturally occurring riboswitches as well as with other regulatory active aptamers. This knowledge may guide the future design of novel synthetic riboswitches for targeted in vivo applications. Structure of the N1–ligand complex—the “OFF”-state of the riboswitch: N1 represses gene expression upon binding to either neomycin B or the closely related but smaller ribostamycin. NMR spectra of N1 bound to either ligand (Supporting Information Figure S1) indicate that both complexes are formed with similarly high affinity and display a high degree of structural similarity suggesting that the contribution of ring IV of neomycin to the interaction is negligible. Thus, we determined the structure of the N1–ribostamycin complex, because of its superior spectral resolution for the ligand resonances, by NMR spectroscopy (see Table 1). Chemical shift assignments and coordinates have been deposited (BMRB code: 16609, pdb-code: 2kxm). The structure of ribostamycin-bound N1 consists of a continuous helical stem with canonical stacking interactions between the G5:C23 and the G9:C22 base pair despite the presence of a flexible three-nucleotide bulge (C6–U8) and a compactly folded apical hexaloop organized around a U-turn motif (U14–A16) closed by the U13:U18 base pair (Figure 1c–e). Ribostamycin rings I and II are sandwiched between the N1 major groove, in the region from G5:C23 to U13:U18 and A17 protruding from the apical loop (Figure 2). Ring III is located close to the backbone of the 3’-strand (U18 to G20). Simultaneous contacts of the ligand with the G5:C23 base pair below and G9:C22 above the bulge (Figure 2b) clamp together the lower and upper helical stem and thus enforce the uninterrupted coaxial helical stacking across the flexible C6–U8 internal bulge. The bulge itself is not interacting with the ligand. A detailed structural description of the N1– ribostamycin complex is given in the Supporting Information. A comparison of the N1–ribostamycin complex with other aminoglycoside binding RNAs reveals partial similarities to known aminoglycoside binding sub-motifs: The helical stem centered at the U10:U21 base pair is similar to the ribosomal [*] Dr. E. Duchardt-Ferner, S. R. Schmidtke, Prof. Dr. J. W hnert Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany) Fax: (+49)69-798-29527 E-mail: woehnert@bio.uni-frankfurt.de

Deep sequencing-based identification of small non-coding RNAs in Streptomyces coelicolor.

Streptomyces coelicolor is considered the model organism among Gram positive, GC rich bacteria. Its genome has been sequenced but little is known about the occurrence and distribution of small non-coding RNAs in this biotechnologically relevant organism. Using deep sequencing we analyzed the transcriptome at the end of exponential growth, which corresponds to the onset of secondary metabolism. We mapped 193 transcriptional start sites of mRNA genes and identified putative new and alternative open reading frames. We identified 63 non-coding RNAs including 29 cis encoded antisense RNAs, and confirmed expression for 11, most of them being growth-phase dependent. A comparison between the sequencing results and bioinformatic sRNA predictions using Dynalign and RNAz revealed only a small overlap between the different approaches.

A fast and efficient translational control system for conditional expression of yeast genes

A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5′UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression.

Riboswitch engineering — making the all-important second and third steps

Synthetic biology uses our understanding of biological systems to develop innovative solutions for challenges in fields as diverse as genetic control and logic devices, bioremediation, materials production or diagnostics and therapy in medicine by designing new biological components. RNA-based elements are key components of these engineered systems. Their structural and functional diversity is ideal for generating regulatory riboswitches that react with many different types of output to molecular and environmental signals. Recent advances have added new sensor and output domains to the existing toolbox, and demonstrated the portability of riboswitches to many different organisms. Improvements in riboswitch design and screens for selecting in vivo active switches provide the means to isolate riboswitches with regulatory properties more like their natural counterparts.

Synthetic riboswitches — A tool comes of age

Within the last decade, it has become obvious that RNA plays an important role in regulating gene expression. This has led to a plethora of approaches aiming at exploiting the outstanding chemical properties of RNA to develop synthetic RNA regulators for conditional gene expression systems. Consequently, many different regulators have been developed to act on various stages of gene expression. They can be engineered to respond to almost any ligand of choice and are, therefore, of great interest for applications in synthetic biology. This review presents an overview of such engineered riboswitches, discusses their applicability and points out recent trends in their development. This article is part of a Special Issue entitled: Riboswitches.