Membrane targeting proteins are recruited to specific membranes during cell signaling events, including signals at the leading edge of chemotaxing cells. Recognition and binding to specific lipids play a central role in targeting reactions, but it remains difficult to analyze the molecular features of such protein-lipid interactions. We propose that the surface diffusion constant of peripheral membrane-bound proteins contains useful information about protein-lipid contacts and membrane dynamics. To test this hypothesis, we use single-molecule fluorescence microscopy to probe the effects of lipid binding stoichiometry on the diffusion constants of engineered proteins containing one to three pleckstrin homology domains coupled by flexible linkers. Within error, the lateral diffusion constants of these engineered constructs are inversely proportional to the number of tightly bound phosphatidylinositol-(3,4,5)-trisphosphate lipids. The same trend is observed in coarse-grained molecular dynamics simulations and hydrodynamic bead calculations of lipid multimers connected by model tethers. Overall, single molecule diffusion measurements are found to provide molecular information about protein-lipid interactions. Moreover, the experimental and computational results independently indicate that the frictional contributions of multiple, coupled but well-separated lipids are additive, analogous to the free-draining limit for isotropic fluids--an insight with significant implications for theoretical description of bilayer lipid dynamics.

RNA-based biosensors and regulatory devices have received significant attention for their potential in a broad array of synthetic biology applications. One of the primary difficulties in engineering these molecules is the lack of facile methods to link sensory modules, or aptamers, to readout domains. Such efforts typically require extensive screening or selection of sequences that facilitate interdomain communication. Bacteria have evolved a widespread form of gene regulation known as riboswitches that perform this task with sufficient fidelity to control expression of biosynthetic and transport proteins essential for normal cellular homeostasis. In this work, we demonstrate that select riboswitch readout domains, called expression platforms, are modular in that they can host a variety of natural and synthetic aptamers to create novel chimeric RNAs that regulate transcription both in vitro and in vivo. Importantly, this technique does not require selection of device-specific “communication modules” required to transmit ligand binding to the regulatory domain, enabling rapid engineering of novel functional RNAs.

Direct sensing of intracellular metabolite concentrations by riboswitch RNAs provides an economical and rapid means to maintain metabolic homeostasis. Since many organisms employ the same class of riboswitch to control different genes or transcription units, it is likely that functional variation exists in riboswitches such that activity is tuned to meet cellular needs. Using a bioinformatic approach, we have identified a region of the purine riboswitch aptamer domain that displays conservation patterns linked to riboswitch activity. Aptamer domain compositions within this region can be divided into nine classes that display a spectrum of activities. Naturally occurring compositions in this region favor rapid association rate constants and slow dissociation rate constants for ligand binding. Using X-ray crystallography and chemical probing, we demonstrate that both the free and bound states are influenced by the composition of this region and that modest sequence alterations have a dramatic impact on activity. The introduction of non-natural compositions result in the inability to regulate gene expression in vivo, suggesting that aptamer domain activity is highly plastic and thus readily tunable to meet cellular needs.

SUMMARY The ZTP riboswitch is a widespread family of regulatory RNAs that upregulate de novo purine synthesis in response to increased intracellular levels of ZTP or ZMP (AICAR). As an important intermediate in purine biosynthesis, ZMP also serves as a proxy for the concentration of 10-formyltetrahydrofolate, a key component of one carbon metabolism. Here we report the structure of the ZTP riboswitch bound to ZMP at a resolution of 1.80 Å. The RNA contains two subdomains brought together through a long-range pseudoknot further stabilized through helix-helix packing. ZMP is bound at the subdomain interface of the RNA through a set of interactions with the ligand's base, ribose sugar and phosphate moieties. Unique to nucleobase recognition by RNAs, the Z base is inner sphere coordinated to a magnesium cation bound by two backbone phosphates. This interaction, along with steric hindrance by the backbone, imparts specificity over related analogs such as ATP/AMP.

Over the past decade the purine riboswitch, and in particular its nucleobase-binding aptamer domain, has emerged as an important model system for exploring various aspects of RNA structure and function. Its relatively small size, structural simplicity and readily observable activity enable application of a wide variety of experimental approaches towards the study of this RNA. These analyses have yielded important insights into small molecule recognition, co-transcriptional folding and secondary structural switching, and conformational dynamics that serve as a paradigm for other RNAs. In this article, the current state of understanding of the purine riboswitch family is examined and how this growing knowledge base is starting to be exploited in the creation of novel RNA devices.