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Jun 17, 2016 - A Hat Trick of New RNA Structures. Jason G. Underwood. Structured RNAs play diverse functional roles in the cell and even catalyze ...
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SYNTHESIS PLATFORM ENABLES DISCOVERY OF MACROLIDE ANTIBIOTICS

Reprinted from Cell, Merk, et al., Breaking Cryo-EM Resolution Barriers to Facilitate Drug Discovery, DOI: 10.1016/ j.cell.2016.05.040. Copyright 2016, with permission from Elsevier.

Adapted by permission from Macmillan Publishers Ltd.: Nature, Myers, et al. 533, 338−345, copyright 2016.

Since its discovery in 1949, erythromycin has become a workhorse antibiotic for treating respiratory and skin infections caused by Streptococcus and Staphylococcus bacteria, among others. Because erythromycin is unstable in the digestive tract, limiting its application in oral treatments, pharmaceutical companies developed more effective erythromycin derivatives. These derivatives are generally synthesized by modifying erthryomycin obtained by fermentation, a process which produces the parrent compound on a ton scale. Despite the availability of the starting material, erythromycin derivatives can be very challenging to make; for example, the synthesis of solithromycin, an advanced clinical candidate effective against a broad range of Gram-positive bacteria, requires 16 consecutive chemical transformations beginning with erythromycin. Morever, development of further erythromycin derivatives is confined to manipulations of the reactive functional groups present in the parent molecule, which can be difficult to target selectively. Mindful of these limitations as well as motivated by the ominous increase in antibiotic-resistant pathogenic bacteria, a research team led by Andrew G. Myers of Harvard University has developed a new platform for discovering novel macrolide antibiotics (Nature 2016, 533, 338−345). Macrolides, a class of molecules including erythromycin, are macrocyclic lactones typically consisting of 14- to 16-membered rings. The research team, including first authors Ian B. Seiple and Ziyang Zhang, focused on linking together readily available building blocks to form molecular fragments, which were then convergently assembled into novel macrolide structures. The piece-wise approach allowed the researchers to append various side chains and introduce heteroatom substitutions in a site-specific manner, ultimately resulting in the gram-scale synthesis of over 300 14and 15-membered ring antibiotic candidates. Several of these macrolides were found to be potent against multiple pathogenic bacteria, including methicillin-resistant Staphylococcusaureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The authors anticipate that their convergent-synthesis platform can be applied to the development of libraries of other classes of antibiotics. Heidi A. Dahlmann © 2016 American Chemical Society

CRYO-EM: IMAGING SMALLER PROTEINS AT HIGHER RESOLUTION

The three-dimensional structures of biological macromolecules have been determined for decades by X-ray crystallography. In this technique, a crystalline sample is bombarded with a beam of X-rays; by analyzing the angle and intensity of the diffracted beams, researchers can construct a map of the electron density in the sample. The better the resolution of the map, the higher the certainty with which the precise locations of individual atoms as well as the nature of their covalent bonds can be determined within the macromolecule. X-ray crystallography is not well-suited for studying proteins that resist crystallization, such as those that are normally found embedded in cell membranes or that are highly dynamic. These limitations have driven the development of cryoelectron microscopy (cryo-EM), in which samples of biomacromolecular complexes are flash-frozen into a thin layer of vitreous (liquid-like) ice. In a manner roughly analgous to light microscopy, the frozen samples are exposed to a beam of electrons, generating two-dimensional projection images of thousands of identical copies of the molecular complex. These images are then computationally combined to create 3D structures. The resolution obtainable by cryo-EM has long lagged behind that of X-ray crystallography, and its application has generally been limited to relatively large (>200 kDa) proteins. However, a team of researchers led by Sriram Subramaniam have recently shattered these paradigms, reporting the 1.8-Å-resolution cryoEM structure of glutamate dehydrogenase (GDH), the first cryoEM structure with sub-2-Å resolution, as well as the cryo-EM structure of the 93 kDa isocitrate dehydrogenase (IDH), the first cryo-EM structure of a sub-100 kDa protein (Cell 2016, http:// dx.doi.org/10.1016/j.cell.2016.05.040). The research team attributes their barrier-breaking successes to judicious sample selection, improved detector performance, and correction for beam-induced specimen movement. They anticipate that further hardware and software developments will enable routine cryo-EM on metabolic proteins, many of which are below 150 kDa. Heidi A. Dahlmann Published: June 17, 2016 1468

DOI: 10.1021/acschembio.6b00494 ACS Chem. Biol. 2016, 11, 1468−1470

ACS Chemical Biology





TRANSLATION IN CELLS TRACKED IN REAL-TIME

Spotlight

A HAT TRICK OF NEW RNA STRUCTURES

From Morisaki et al., Science, 2016, DOI: 10.1126/science.aaf0899. Reprinted with permission from AAAS.

When it comes to understanding living systems, the ability to quantify biological processes occurring in real time in cells is a

Reprinted from Mol. Cell, Sharma et al., Global Mapping of Human RNA−RNA Interactions, DOI: 10.1016/j.molcel.2016.04.030. Copyright 2016, with permission from Elsevier.

major research goal. For years, scientists have been able to quantify the transcription of DNA to RNA at single gene resolution in cells; now, Tatsuya Morisaki and co-workers

Structured RNAs play diverse functional roles in the cell and even catalyze fundamental processes including translation and RNA splicing. Textbooks usually depict mRNAs as unstructured strings, but this may be due to the difficulty challenge of understanding RNA structure on a global level. Recent advances in high throughput sequencing made probing the transcriptome more feasible, and numerous methodologies emerged, including proximity ligation, a tool pioneered for understanding long-range interactions of the genome. Now, a series of three studies published simultaneously introduce a clever improvement to proximity ligation methods. The groups utilized a classic technique of the RNA biochemist, in vivo psoralen cross-linking, prior to RNA fragmentation. After ligating proximal RNA fragments and converting to cDNA, short tag sequencing identified chimeric molecules indicative of RNA− RNA interactions in the cell. After validating on known noncoding RNA structures and interactions, the studies delve in global RNA interactions, but each study focuses on a different aspect of the data. Aw et al. (Mol. Cell, DOI: 10.1016/ j.molcel.2016.04.028) combined their mRNA structural data with ribosome profiling data to show that 5′ mRNA structures reduce translation efficiency, while long-range interactions between the 5′ and 3′ ends of an mRNA lead to higher translation efficiency. They also show that sometimes human mRNAs encoding broadly similar cellular modules (e.g., transcription) physically interact with one another in trans. Sharma et al. (Mol. Cell, DOI: 10.1016/j.molcel.2016.04.030) used the data to identify several snoRNA-mRNA interactions and then demonstrated that availability of the snoRNA actually affects target mRNA levels. Finally, Lu et al. (Cell, DOI: 10.1016/j.cell.2016.04.028) employed human and mouse cells in parallel and used their data to understand the conservation and covariation behind long-range cis RNA stuctures. The researchers also showed that alternative structures are common in RNAs, as are RNA−RNA interactions in trans. They take on a formidable task, using their structural information to tackle Xist, a 19-kb noncoding RNA critical for X chromosome inactivation. The good news for researchers is that the three parallel studies introduce complementary methods and unleash very large data sets to help understand RNA structure. The bad news is that three new sequencing acronyms enter the lexicon in parallel,

have reported a corresponding method for following translation of RNA to protein (Science 2016, DOI: 10.1126/ science.aaf0899). To visualize translation in real time, the team developed a system in which a newly synthesized protein as well as the mRNA from which it was being translated could be labeled simultaneously. The team designed a plasmid in which the gene for a protein of interest (POI) was flanked on one side by DNA encoding an epitope tag and on the other side by a DNA sequence that would be transcribed to form specific RNA structures known as MS2 stem-loop repeats. Upon introduction of the plasmid into cells, the plasmid construct would be transcribed into an mRNA strand containing the MS2 stem-loop repeats, which would bind to fluorescently labeled MS2 coat protein (MCP) and thus allow individual mRNA strands to be visualized. Subsequent translation of the labeled mRNA would produce the epitope-tagged POI, which would bind to corresponding fluorescently labeled antibodies and enable the POIs to be visualized as they were being synthesized. Using this method, dubbed nascent chain tracking, Stasevich and co-workers determined that ribosomes elongated peptide chains by approximately 10 amino acids per second and that single mRNA chains could contain multiple ribosomes, which cluster into groups called polysomes, at a density of about one ribosome per 200−900 nucleotides. The imaging technique also revealed that polysomes formed globular rather than linear clusters and that a small fraction of polysomes actually processed more than one mRNA strand at a time. Heidi A. Dahlmann 1469

DOI: 10.1021/acschembio.6b00494 ACS Chem. Biol. 2016, 11, 1468−1470

ACS Chemical Biology

Spotlight

SPLASH, LIGR-seq, and PARIS. Check out these three studies to decode and learn. Jason G. Underwood



A SYNTHETIC ANION-π ENZYME

Reprinted with permission from Cotelle et al., ACS Cent. Sci., DOI: 10.1021/acscentsci.6b00097. Copyright 2016 American Chemical Society.

Catalysts that employ novel interactions could expand the chemical toolbox, offering new ways to generate molecules with high enantio- and chemoselectivity. Anion−π interactions between negatively charged species and electron-poor π systemsare an intriguing, newly investigated counterpoint to canonical cation−π interactions. Now Cotelle et al. have designed an innovative synthetic anion−π enzyme that catalyzes the addition of malonic acid half thioesters (MAHT) to enolate acceptors (ACS Cent. Sci. 2016, DOI: 10.1021/acscentsci.6b00097). Biosynthesis starts with this fundamental reaction, and these carbon−carbon bond forming processes are critical components in the construction of polyketide natural products. But this reaction requires an enzyme catalyst. Cotelle et al. wanted to stabilize the planar MAHT enolate using a catalytic “active site” triad that included an electron-poor polyaromatic surface linked to a base and a biotin molecule. They used the biotin to attach this synthetic catalyst to streptavidin. They screened a series of different triads that varied the linker from the biotin moiety, the size and electron density of the polyaromatic core, and the steric bulk around the base. As the team screened the various options, they found only one triad that showed any enantioselectivity (10% ee), but when they tested a range of pH conditions, they found that at pH 3.0, they could obtain product with 41% ee and a chemoselectivity of more than 30. They then screened a series of streptavidin mutants and achieved perfect chemoselectivity and 95% ee with S112Y and S112F mutants, suggesting that the additional π interactions played an important role in the enhanced performance. Kinetic studies showed that faster reactions were more selective, showing that stabilizing the transition state increased the enantioselectivity. K121 in streptavidin was also critical for activity and stereoselectivity. Docking simulations of the mutant streptavidin protein with the triad provide a clear visual picture of how this synthetic enzyme catalyzes this reaction. This first artificial anion−π enzyme is more effective than existing organocatalysts, and these results suggest other opportunities for expanding this strategy or adapting other unorthodox chemical recognition modes to produce new catalysts. Sarah A. Webb

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DOI: 10.1021/acschembio.6b00494 ACS Chem. Biol. 2016, 11, 1468−1470