Synapses are critical targets of Alzheimer's disease (AD), a highly prevalent neurodegenerative disease associated with accumulation of extracellular amyloid-β peptides. Although amyloidosis and aggregation of the 42-amino acid amyloid-β (Aβ 42 ) have long been considered pathogenic triggers for AD, clinical evidence linking high levels of Aβ 42 with normal cognition challenges this hypothesis. To resolve this conundrum on the role of Aβ 42 in regulating synaptic activity, we used an adeno-associated viral vector approach that triggers extracellular accumulation of Aβ 42 and spatial memory impairment. We show that Aβ 42 leads to an early increase in excitatory and proximal inhibitory synaptic transmission onto hippocampal CA1 pyramidal cells, and an increased expression of the glutamate transporter GLT-1 in these cells. Aβ 42 accumulation does not cause early cognitive deficits unless accompanied by an increased neuronal GLT-1 expression, suggesting this transporter is a critical mediator of Aβ 42 's effects. These findings unveil key molecular and cellular mechanisms implicated with AD pathogenesis.

Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting glutamate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes, and previous estimates have been obtained about their surface expression in astrocytes of the rat hippocampus and cerebellum. Analogous estimates for the mouse hippocampus are currently not available. In this work, we derive the surface density of astrocytic glutamate transporters in mice of different ages via quantitative dot blot. We find that the surface density of glial glutamate transporters is similar in 7-8 week old mice and rats. In mice, the levels of glutamate transporters increase until about 6 months of age and then begin to decline slowly. Our data, obtained from a combination of experimental and modeling approaches, point to the existence of stark differences in the density of expression of glutamate transporters across different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on their level of synaptic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on how heterogeneity in the spatial distribution of glutamate transporters in the plasma membrane of hippocampal astrocytes my alter glutamate receptor activation out of the synaptic cleft.

Glutamate transporters preserve the spatial specificity of synaptic transmission by limiting gluta-mate diffusion away from the synaptic cleft, and prevent excitotoxicity by keeping the extracellular concentration of glutamate at low nanomolar levels. Glutamate transporters are abundantly expressed in astrocytes. Previous estimates in the rat hippocampus suggest that the surface density of glutamate transporters in astrocytic membranes is ~ 10, 800 μm−2. Here, we estimate their surface density in astrocytic membranes of the mouse hippocampus, at different ages. By using realistic 3D Monte Carlo reaction-diffusion models, we show that varying the local glutamate transporter expression in astrocytes can alter profoundly the activation of extrasynaptic AMPA and NMDA receptors. Our findings show that the average density of astrocyte membranes and their surface density of glutamate transporters is higher in mice compared to rats, and increases with mouse age. There are stark differences in the density of expression of these molecules in different sub-cellular compartments, indicating that the extent to which astrocytes limit extrasynaptic glutamate diffusion depends not only on the level of astrocytic coverage, but also on the identity of the astrocyte compartment in contact with the synapse. Together, these findings provide information on the spatial distribution of glutamate transporters in the mouse hippocampus, which can be used in mathematical models of the spatiotemporal profile of extracellular glutamate after synaptic release.The astrocyte membrane density in the hippocampal neuropil is higher than in rats.The surface density of glutamate transporters in mouse astrocytes is higher than in rats and varies widely across different sub-cellular compartments.The identify of the astrocyte compartment in contact with a synapse, not only the extent of astrocytic coverage, is a main determinant of glutamate spillover and extrasynaptic receptor activation.

The Cover Feature shows 20 of 200 compounds that were bound strongly in silico within an RNA grove. The 200 compounds were selected from 305,000. They were used to target an RNA function, the tRNA-dependent regulatory mechanism unique to Gram-positive pathogens, in search of a novel compound that inhibits bacterial growth and reduces emergent resistance. The green RNA helix (top and bottom) is part of Stem 1 of the 5’-untranslated region of nascent mRNAs, the transcription of which is regulated by unacylated tRNA. The blue is a loop that consists of an adenosine-rich stack and a red “codon” that binds the tRNA anticodon. More information can be found in the Full Paper by Paul F. Agris et al. on page 758 in Issue 7, 2019 (DOI: 10.1002/cmdc.201800744).

The emergence of multidrug-resistant bacteria necessitates the identification of unique targets of intervention and compounds that inhibit their function. Gram-positive bacteria use a well-conserved tRNA-responsive transcriptional regulatory element in mRNAs, known as the T-box, to regulate the transcription of multiple operons that control amino acid metabolism. T-box regulatory elements are found only in the 5 '-untranslated region (UTR) of mRNAs of Gram-positive bacteria, not Gram-negative bacteria or the human host. Using the structure of the 5 ' UTR sequence of the Bacillus subtilis tyrosyl-tRNA synthetase mRNA T-box as a model, in silico docking of 305 000 small compounds initially yielded 700 as potential binders that could inhibit the binding of the tRNA ligand. A single family of compounds inhibited the growth of Gram-positive bacteria, but not Gram-negative bacteria, including drug-resistant clinical isolates at minimum inhibitory concentrations (MIC 16-64 mu g mL(-1)). Resistance developed at an extremely low mutational frequency (1.21x10(-10)). At 4 mu g mL(-1), the parent compound PKZ18 significantly inhibited in vivo transcription of glycyl-tRNA synthetase mRNA. PKZ18 also inhibited in vivo translation of the S. aureus threonyl-tRNA synthetase protein. PKZ18 bound to the Specifier Loop in vitro (K-d approximate to 24 mu m). Its core chemistry necessary for antibacterial activity has been identified. These findings support the T-box regulatory mechanism as a new target for antibiotic discovery that may impede the emergence of resistance.

Most animal species operate according to a 24-h period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCNmodulates hippocampal-dependent memory, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we identify cell-type-specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors. Astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation, and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampaldependent learning in vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings highlight important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus, and alter the temporal dynamics of cognitive processing.

tRNA‐dependent control of gene expression, T‐boxes, are RNA regulatory elements located at the 5′‐untranslated region (5′‐UTR) in numerous gram‐positive bacteria. T‐boxes control the expression of several genes involved in tRNA aminoacylation, as well as amino acid metabolism and transport. T‐boxes function by recognizing the aminoacylation status of their cognate tRNA ligand: uncharged tRNA can stabilize an antiterminator complex through an acceptor stem – antiterminator interaction, thus allowing transcription of the downstream operon. Aminoacylated tRNA cannot form this interaction, therefore the thermodynamically more stable terminator hairpin is formed, causing transcription to halt. Given that T‐boxes are found exclusively in gram‐positive bacteria and share highly conserved regions necessary for tRNA binding, this RNA regulatory element represents a prime target for antibacterial drug development. Docking studies using atomic‐resolution T‐box structures identified several potential candidates. 43 putative antibacterial compounds representing distinct chemical families were selected for further characterization. Antibacterial microdilution studies resulted in a moderately active hit compound (PKZ18) that has been selected for further antibacterial drug development. Characterization studies demonstrate that PKZ18 is highly refractory to resistance, displays low cytotoxicity, and possesses activity against a wide range of gram‐positive bacteria, including clinical isolates of Staphylococcus aureus, Streptococcus pyogenes, and Clostridium difficile. Mechanism of action studies confirm binding of PKZ18 to both glycine and tyrosine T‐boxes, as well as inhibition of threonine tRNA synthetase, a protein under the control of a threonine T‐box in S. aureus. Structure‐activity relationship (SAR) studies of PKZ18 have identified the essential chemical moieties for both specificity as well as activity. Hit‐to‐lead optimization of PKZ18 by medicinal chemistry and further SAR studies is on‐going.

Assembly of HIV-1 viral particles is a critical step of the HIV-1 life cycle; yet many details of this complex process are unknown. The Gag polyprotein drives viral particle assembly at the plasma membrane via three different types of interactions: protein-protein, protein-RNA, and protein-membrane interactions. As an approach to tease apart the importance of these interactions during viral particle assembly, in particular at the step of Gag membrane binding, we have developed an in vitro liposome-binding assay. Below we describe how to prepare liposomes, which serve as model membranes, and how to assess their interaction with Gag by liposome flotation centrifugation. Additionally, we outline extensions of this basic assay that can be used to address the role of RNA in regulating Gag-membrane interactions.

HIV-1 particle assembly, which occurs at the plasma membrane (PM) of cells, is driven by the viral polyprotein Gag. Gag recognizes phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific phospholipid, via the highly basic region (HBR) in its N-terminal matrix (MA) domain. The HBR is also known to bind to RNA. We have previously shown, using an in vitro liposome binding assay, that RNA inhibits Gag binding to membranes that lack PI(4,5)P2 If this RNA block is removed by RNase treatment, Gag can bind nonspecifically to other negatively charged membranes. In an effort to identify the RNA species that confer this inhibition of Gag membrane binding, we have tested the impact of purified RNAs on Gag interactions with negatively charged liposomes lacking PI(4,5)P2 We found that some tRNA species and RNAs containing stem-loop 1 of the psi region in the 5' untranslated region of the HIV-1 genome impose inhibition of Gag binding to membranes lacking PI(4,5)P2 In contrast, a specific subset of tRNAs, as well as an RNA sequence previously selected in vitro for MA binding, failed to suppress Gag-membrane interactions. Furthermore, switching the identity of charged residues in the HBR did not diminish the susceptibility of Gag-liposome binding for each of the RNAs tested, while deletion of most of the NC domain abrogates the inhibition of membrane binding mediated by the RNAs that are inhibitory to WT Gag-liposome binding. These results support a model in which NC facilitates binding of RNA to MA and thereby promotes RNA-based inhibition of Gag-membrane binding.

T‐box riboswitches are RNA regulatory elements located at the 5′‐untranslated region (5′‐UTR) found in numerous gram‐positive bacteria that control several genes involved in tRNA aminoacylation, as well as amino acid metabolism and transport. T‐boxes function by recognizing the charge status of their cognate tRNA ligand: uncharged tRNA stabilizes an antiterminator complex when bound, allowing transcription of the downstream operon, whereas charged tRNA is unable to fully bind the T‐box, allowing a terminator loop to persist and halt transcription. Given that T‐boxes are found exclusively in gram‐positive bacteria and share highly conserved regions necessary for tRNA binding, this RNA regulatory element represents a prime target for antibacterial drug development. Using docking studies to known atomic‐resolution T‐box structures, 43 putative antibacterial compounds representing distinct chemical families were selected for further characterization. Antibacterial microdilution studies resulted in a moderately active hit compound (PKZ18) that has been selected for antibacterial drug development. Characterization studies demonstrate that PKZ18 is refractory to resistance, displays low cytotoxicity, and possesses activity against a wide range of gram‐positive bacteria, including clinical isolates of Staphylococcus aureus , Streptococcus pyogenes , and Clostridium difficile . Mechanism of action studies confirm binding of PKZ18 to both glycine and tyrosine T‐boxes. Structure‐activity relationship (SAR) studies have revealed essential moieties for both selectivity as well as activity. Hit‐to‐lead optimization of PKZ18 by medicinal chemistry and further SAR studies is on‐going.

Epitranscriptomics is the study of global modification patterns to both coding and noncoding RNA. Understanding the epitranscriptomic profile of disease states or individual patients is imperative to understanding human health and molecular disease pathology. Modifications have long been established as important determinants of tRNA stability, dynamics, and ribosome binding and of maintenance of the translational reading frame. These modifications also serve as biomarkers for several human diseases, including type 2 diabetes, cardiac dysfunction, intellectual disability, and skin, breast, and colorectal cancers. Of particular note, several mitochondrial disorders trace their molecular pathogenesis to deficiencies in specific tRNA modifications. Pathology can also be attributed to mutations affecting protein recognition of tRNA substrates. However, protein recognition of RNA modification is at present an underdeveloped field and the subject of increasing attention. Epitranscriptomic profiling will be readily achievable with new advances in the detection of RNA modifications by peptides and mass spectrometry at the attomole level. These technologies will allow for single-cell analysis of modifications and will serve as a platform for increased sensitivity for biomarker identification. Thus, RNA modifications are a real-time code to RNA structure and function that has yet to be deciphered.

T‐boxes are gram positive specific riboswitches that sense the charged status of tRNAs to regulate­ transcription of operons containing corresponding tRNA synthetase, amino acid metabolism and amino acid uptake genes. An uncharged tRNA spans two structural elements of the T‐box. The 5′ specifier loop nucleotides are complementary to the anticodon of a specific tRNA, and an antiterminator domain binds to the uncharged 3′ CCA arm. The latter interaction cannot form when a tRNA is charged, leading to the formation of an alternate transcription termination hairpin. T‐boxes can be found in most gram positive species, often regulating multiple operons (up to 19) with different tRNAs. Redundant use of T‐boxes and specificity of this element to gram positive bacteria make T‐boxes an excellent target for the development of small molecule, broad spectrum antibiotics with a low probability for antibiotic resistance development. Our lab has identified a small molecule compound that interacts with the specifier loop in silico, selectively kills gram positive bacteria and is non‐toxic to human cells. We have also characterized the Mg2+‐dependent interaction between tRNAGly and the B. subtilis glyQS 5′ UTR T‐box element using gel shift assays and isothermal titration calorimetry. These assays will be extended to elucidate the mechanism of small molecule inhibition of T‐box regulation in vitro.

embrane binding of HIV-1 Gag is one of the essential stepsinvirusassembly,whichtakesplaceprimarilyattheplasmamembrane(PM)(1).Thematrix(MA)domainofGagisessentialfor directing virus assembly specifically to the PM. MA has anN-terminalmyristoylmoietythatfacilitateshydrophobicinterac-tionofGagwithmembranes(2,3).Thesecondsignalrequiredforefficient association of Gag with membranes is the highly basicregion (HBR) in MA, which spans residues 17 to 31 of MA. TheHBRmediatestheelectrostaticinteractionwithcellularacidiclip-ids (4–10), in particular, a PM-specific phospholipid, phosphati-dylinositol-(4,5)-bisphosphate[PI(4,5)P

The matrix domain promotes plasma-membrane-specific binding of HIV-1 Gag through interaction with an acidic lipid phosphatidylinositol-(4,5)-bisphosphate. In in vitro systems, matrix-bound RNA suppresses Gag interactions with phosphatidylserine, an acidic lipid prevalent in various cytoplasmic membranes, thereby enhancing the lipid specificity of the matrix domain. Here we provide in vitro and cell-based evidence supporting the idea that this RNA-mediated suppression occurs in cells and hence is a physiologically relevant mechanism that prevents Gag from binding promiscuously to phosphatidylserine-containing membranes.

Translational bypassing is a unique phenomenon of bacteriophage T4 gene 60 mRNA wherein the bacterial ribosome produces a single polypeptide chain from a discontinuous open reading frame (ORF). Upon reaching the 50-nucleotide untranslated region, or coding gap, the ribosome either dissociates or bypasses the interruption to continue translating the remainder of the ORF, generating a subunit of a type II DNA topoisomerase. Mutational and computational analyses have suggested that a compact structure, including a stable hairpin, forms in the coding gap to induce bypassing, yet direct evidence is lacking. Here we have probed the secondary structure of gene 60 mRNA with both Tb³⁺ ions and the selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) reagent 1M7 under conditions where bypassing is observed. The resulting experimentally informed secondary structure models strongly support the presence of the predicted coding gap hairpin and highlight the benefits of using Tb³⁺ as a second, complementary probing reagent. Contrary to several previously proposed models, however, the rest of the coding gap is highly reactive with both probing reagents, suggesting that it forms only a short stem-loop. Mutational analyses coupled with functional assays reveal that two possible base-pairings of the coding gap with other regions of the mRNA are not required for bypassing. Such structural autonomy of the coding gap is consistent with its recently discovered role as a mobile genetic element inserted into gene 60 mRNA to inhibit cleavage by homing endonuclease MobA.

Nucleic acid higher order structure is of intense interest in antisense and antigene strategies toward novel chemotherapeutic agents. Understanding how structural characteristics affect solution-phase properties is essential for a rational approach to nucleic acid-targeted drug design. The most dominant nucleic acid secondary structure is the hairpin, formed by intrastrand hydrogen bonding between complementary nucleobases. We have previously applied gas-phase hydrogen/deuterium exchange (HDX) with mass spectrometry detection to show that anionic DNA duplexes have lower HDX rates than their constituent monomers, indicating that hydrogen bonding can shield hydrogens from exchanging with the bath gas D(2)S. The same HDX assay is applied here to investigate nucleic acid hairpin structure. Variations in hairpin solution-phase stabilities are achieved by changing their loop size, stem length, and stem composition (ratio of G/C and A/T(U) base pairs in the stem). These differences can be carried into the gas phase because electrospray ionization is a gentle ionization method that is able to preserve noncovalent interactions. Observed gas-phase HDX rates of these hairpins are consistent with their relative solution-phase stabilities as predicted by MFold, i.e., less stable nucleic acid hairpins exchange faster than more stable hairpins. To our knowledge, the presented experiments demonstrate for the first time that gas-phase HDX may be used to characterize nucleic acid higher order structure and the results suggest that the relative stabilities of nucleic acid hairpins in the gaseous phase are correlated with those in solution.

In the last several years RNA has attracted so much scientific attention that it seems hard to believe (especially to those of us who learned about catalytic RNAs from textbooks) that there was a time when RNA was the stepchild of biochemists—not stable enough to store genetic information and not structurally and functionally diverse enough to catalyze reactions. From today's perspective, it is therefore impossible to fully appreciate the intellectual merit of the breathtaking proposal, put forward in parallel by Orgel, Woese, and Crick in 1968,1-3 that an RNA world could provide a simple solution to the old chicken-and-egg problem of evolution—which came first, the DNA to encode proteins or the proteins required to synthesize DNA?—as RNA might be able to perform both functions. One way to appreciate the forward-thinking nature of this proposal is to consider the fact that it took another 15 years of scientific research before the first evidence for catalytic RNA was documented. One early finding that suggested RNA might be able to exert catalytic action was Aaron Klug's determination of the crystal structure of tRNA in 1974.4 This structure exemplified the intricate and compact tertiary structures that RNA is capable of adopting, a key feature of proteins that are required for catalytic activity. Another 8 years later Cech and coworkers, as well as Altman and coworkers, finally stumbled upon catalytic RNAs involved in self-splicing and tRNA maturation, respectively.5, 6 The unveiling of catalytic RNA has transformed the scientific community's perception of RNA and has led to the birth of an entire new field devoted to the study of the structure and function of RNA. Numerous examples of catalytic RNAs have since been found to function in processes such as viral replication (HDV, hammerhead, hairpin, and other small ribozymes7-9), protein translation,10 and the maturation of tRNAs (RNase P6), rRNAs (RNase MRP11), and mRNAs (self-splicing introns5, 12, 13 and possibly the spliceosome,14, 15 itself a Nobel-winning discovery by Phil Sharp and Richard Roberts). As a result we now have a fairly sophisticated understanding of RNA's capabilities and limitations. The mechanism of some RNA enzymes is understood as well as or some may say in even greater detail than that of many protein enzymes.16-19 In addition, we now have atomic resolution structures of many RNA molecules,20-28 including the ribosome.29-32 Such information has allowed us to make significant progress in determining the details of the cellular role of many RNA molecules, including the cell's largest RNA machine, the ribosome. This progress by itself is impressive and would have been unthinkable 25 years ago, when the Cech and Altman labs were preparing their seminal publications. However, more recently there has been yet another RNA revolution: In 1998, Fire and Mello reported that double-stranded RNA could strongly and specifically repress gene expression in worms.33 They had uncovered the RNAi pathway, for which they were awarded the most recent Nobel price in Medicine. Their groundbreaking discovery has proven to be one of the most exiting new frontiers in RNA research, as well as an invaluable “genetic” tool for the study of higher eukaryotic organisms. This discovery was also the first insight into how RNA is used in biology to regulate gene expression in a spatial and temporal manner via targeting of the mRNA through complementary base-pair interactions. A related regulatory role was uncovered when Kadner's and Soberon's labs reported the existence of metabolite-sensitive structures in the 5′-regulatory regions of individual mRNAs in bacteria.34, 35 These types of RNA sequences, now dubbed “riboswitches,” have been extensively characterized by the Breaker lab at Yale (e.g., Refs.36-39). Breaker and coworkers have shown that metabolite binding induces conformational rearrangements in the RNA, which can regulate transcription or translation of mRNAs. In addition, it is now clear that some riboswitches are evolutionarily conserved and coregulate entire metabolic pathways in response to intermediates in that pathway. It was thus fitting that Ron Breaker gave the keynote address at the Ninth Annual Michigan RNA Society meeting held in April 2007 at the University of Michigan campus in Ann Arbor. In his talk he gave a beautiful summary of this field, which has been almost single-handedly unraveled by his lab. He and his coworkers have now uncovered 16 classes of riboswitches,40 explored the folding of the RNA in the presence and absence of the metabolite (e.g., Ref.41, see Figure 1), and mapped the biosynthetic pathways regulated via these riboswitches.38, 40 Bioinformatics tools have been invaluable for these discoveries and have been aided by the simplicity of RNA secondary structure prediction, given that phylogenetic information is available from genome sequencing. Thus, the beauty of Breaker's work is that experiments and computation go hand in hand. In an exciting new frontier, Breaker also reported the discovery of metabolite-sensitive regulatory elements that affect alternative splicing in fungi.42 In addition to this tour de force talk, three posters from the Walter lab at the University of Michigan, as well as one from the Walton lab at Michigan State explored conformational dynamics of riboswitches and their use as chemical sensors. Shown is an artistic rendering of the 2.05 Å crystal structure of the thiamine pyrophosphate (TPP) riboswitch bound to TPP.44 The structure represents a striking example of an intricately folded RNA element that precisely binds a specific ligand through the exact organization of multiple binding pockets that each recognize and coordinate with a particular defined functional moiety of the ligand. Figure courtesy of Maximilian Bailor. This meeting, however, was by no means devoted solely to riboswitches. Instead, it covered a broad range of RNA biochemistry, biophysics, and biology and included talks and posters on RNA structure and dynamics, the function of ribozymes, analysis of the translation machinery, and ribosome assembly. Highlights from oral presentations include the characterization of the dynamics of the transactivation response element (TAR) from HIV, using the recently pioneered NMR technique of residual dipolar coupling. In his work, which was recognized with an award for the best talk, Max Bailor from the Al-Hashimi lab at the University of Michigan characterized the binding of four closely related aminoglycoside antibiotics to TAR. He was able to show a surprising degree of promiscuity in the recognition of these antibiotics, demonstrating the structural flexibility that is characteristic (for better or for worse) of RNA molecules. Interestingly, the changes in the RNA molecule depend on the chemical modifications of these antibiotics in a modular manner. Additional posters from the Hoogstraten (Michigan State University) and Al-Hashimi labs explored further the use of NMR techniques to analyze RNA's structural dynamics. In related work, Tuhina Banerjee, a postdoctoral researcher from Andrew Feig's lab at Wayne State University, presented an analysis of conformational changes in RNAs as modeled by kissing interactions, which can be found throughout biology, including in HIV viral maturation and in the regulation of gene expression via noncoding RNAs in bacteria. Banerjee used isothermal titration calorimetry and single molecule fluorescence spectroscopy to show that her model hairpins form kissing duplexes unproductively, many times before they eventually melt into long duplexes. This provides insight into the function of proteins that catalyze duplex formation, such as Hfq in bacteria. These proteins are thus predicted to work by lowering the barrier to duplex formation and not by stabilizing the kissing complexes or by lowering the barrier to kissing loop formation. A review by Feig on “Applications of Isothermal Titration Calorimetry in RNA Biochemistry and Biophysics” is included in this issue of Biopolymers. Single molecule experiments are used routinely by the Walter and Rueda (Wayne State) labs to study RNA structure. These labs presented posters discussing conformational heterogeneity in the hairpin ribozyme, loop–loop interactions in the hammerhead ribozyme, and RNA folding using U2 and U6 RNA, which form the active site of the spliceosome. In addition, Miguel Pereira from the Walter lab presented a single-molecule fluorescence resonance energy transfer-based analysis of how kissing loops and the central junction cooperate in a Mg2+-dependent manner to ensure folding of the Varkud satellite(VS) ribozyme from the mitochondrial VS RNA of Neurospora crassa to its catalytically active structure. Pereira's data provide information on the interactions stabilizing the catalytically active fold and will allow further dissection of the role of specifically bound Mg2+-ions as well as structural components in stabilizing this fold. The use of single molecule experiments to unravel catalytic mechanisms of RNA enzymes is reviewed by Walter and coworkers in “Focus on Function: Single Molecule RNA Enzymology”, which is also in this issue of Biopolymers. Ribozymes were also the focus of additional talks and posters from the Hoogstraten, Fierke (University of Michigan) and Engelke labs (University of Michigan). Kristin Smith from the Fierke lab discussed her work on bacterial RNase P holoenzyme, which cleaves the 5′-leader sequence from pre-tRNA molecules. She has dissected the contribution of a conserved charged protein motif, the RNR (arginine–asparagine–arginine) motif, found in the single protein component from bacterial RNase P, on binding of both RNase P RNA as well as pre-tRNA substrate using catalytic activity as a readout. Her work comparing the activities of wild type and mutant holoenzymes indicates that the RNR motif is located at the interface between RNaseP and pre-tRNA and helps to correctly assemble a catalytically active holoenzyme. Mutations in this region weaken binding to RNase P RNA and pre-tRNA substrate and, furthermore, reduce catalytic activity of the assembled RNP. In contrast, work from Scott Walker in Dave Engelke's lab focuses on dissecting the topology of the much more complex yeast RNase P enzyme, which, in addition to the RNA component, contains nine proteins. Walker's work uses a novel crosslinking approach and tagged yeast strains to dissect RNA-RNA and RNA-protein interactions and gain a first handle on this biochemically challenging complex. “RNA-protein interactions in RNase P” are also reviewed by Fierke and colleagues in this issue. Given the importance of ribozymes to the RNA field, this issue of Biopolymers contains two related reviews on catalytic nucleic acids. Charles Hoogstraten gives a broad overview on “Functional strategies of catalytic RNAs and RNPs.” “Recent Advances in DNA Catalysis,” which are taking simplification of the chemical repertoire of catalysts a step further, are reviewed by Scott Silverman (University of Illinois, Urbana Champagne). Toward more complex biological systems, the Feig lab presented work indicating that Hfq, the bacterial Sm-like protein, binds tRNAs with strong affinity, and also interacts with proteins involved in tRNA metabolism. This supports the idea that Hfq might play a hitherto unrecognized role in tRNA metabolism. An outstanding poster on this work was presented by Taewoo Lee and was recognized as the best of over 40 posters. Given the exciting, recent, high-resolution structures of ribosomes, it may not be very surprising that there were many talks aiming to understand the role of modifications as well as dynamics for ribosome function. Work presented from the Chow, Santa Lucia, and Cunningham labs (all at Wayne State) used NMR, chemical biology tools, genetic, and biochemical experiments to address these exciting questions. A talk presented by Tek Lamichhane, from the Cunningham lab, described the use of mutational analysis to tease out the function of base modifications in a conserved loop of the mature 16S rRNA that resides near the ribosomal P-site. Lamichhane's data indicate that a specific modifications improve fidelity by limiting stop-codon read-through and misincorporation of incorrect amino acids into the nascent polypeptide. By combining these functional results with the available structural information, this group has begun making hypotheses as to how the wild-type structure ensures proper protein translation. The recent ribosome structures as well as large-scale mass-spectrometry approaches have provided a strong background and renewed interest in studies on ribosome assembly. Work in this area, presented by the Britton (Michigan State), Maddock and Karbstein labs (both at the University of Michigan), covered the function of GTPases and other assembly factors in bacteria and yeast, respectively. This work has revealed that GTPases are the largest class of essential ribosome assembly factors in bacteria and has started to provide insight into the role of GTP hydrolysis by the yeast GTPase Bms1 in promoting binding of a putative RNA endonuclease to nascent ribosomes. The role of GTPases in ribosome assembly was also reviewed in a recent issue of Biopolymers.43 In summary, the meeting showcased work on every aspect on RNA's function, a repertoire, which is likely to further expand in the future. It is clear that RNA-centered research will remain at the forefront of biological research in Michigan and beyond, and we look forward to new discoveries in the years to come. For more information on the RNA community and RNA meetings refer to http://www.rnasociety.org and http://www.umich.edu/∼superrna. Gabrielle Todd*, Katrin Karbstein , * Chemical Biology Ph.D. Program, University of Michigan, Ann Arbor, MI 48109-1055, Department of Chemistry and Department of Biological Chemistry, University of Michigan, 930 N. University, Ann Arbor, MI 48109-1055.

This chapter contains sections titled: Introduction Application Example Troubleshooting Frontiers in Footprinting Data Analysis References