Physical Sciences and Mathematics Commons^™

Connexin-46/50 In A Dynamic Lipid Environment Resolved By Cryoem At 1.9 Å, Jonathan A. Flores, Bassam G. Haddad, Kimberly A. Dolan, Janette Bernadette Myers, Craig C. Yoshioka, Jeremy Yoshioka, Daniel M. Zuckerman, Steve L. Reichow

Chemistry Faculty Publications and Presentations

Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid …

Size-Dependent Interactions Of Lipid-Coated Gold Nanoparticles: Developing A Better Mechanistic Understanding Through Model Cell Membranes And In Vivo Toxicity, Arek M. Engstrom, Ryan A. Faase, Joe E. Baio, Marilyn R. Mackiewicz, Stacey L. Harper

Chemistry Faculty Publications and Presentations

Introduction: Humans are intentionally exposed to gold nanoparticles (AuNPs) where they are used in variety of biomedical applications as imaging and drug delivery agents as well as diagnostic and therapeutic agents currently in clinic and in a variety of upcoming clinical trials. Consequently, it is critical that we gain a better understanding of how physiochemical properties such as size, shape, and surface chemistry drive cellular uptake and AuNP toxicity in vivo. Understanding and being able to manipulate these physiochemical properties will allow for the production of safer and more efficacious use of AuNPs in biomedical applications.
Methods and Materials: Here, …

Limited Sequence Diversity Within A Population Supports Prebiotic Rna Reproduction, Ryo Mizuuchi, Niles Lehman

Chemistry Faculty Publications and Presentations

The origins of life require the emergence of informational polymers capable of reproduction. In the RNA world on the primordial Earth, reproducible RNA molecules would have arisen from a mixture of compositionally biased, poorly available, short RNA sequences in prebiotic environments. However, it remains unclear what level of sequence diversity within a small subset of population is required to initiate RNA reproduction by prebiotic mechanisms. Here, using a simulation for template-directed recombination and ligation, we explore the effect of sequence diversity in a given population for the onset of RNA reproduction. We show that RNA reproduction is improbable in low …

Multivalency Regulates Activity In An Intrinsically Disordered Transcription Factor, Sarah Clark, Janette B. Myers, Ashleigh King, Radovan Fiala, Jiri Novacek, Grant Pearce, Steve L. Reichow, Elisar J. Barbar

Chemistry Faculty Publications and Presentations

The transcription factor ASCIZ (ATMIN, ZNF822) has an unusually high number of recognition motifs for the product of its main target gene, the hub protein LC8 (DYNLL1). Using a combination of biophysical methods, structural analysis by NMR and electron microscopy, and cellular transcription assays, we developed a model that proposes a concerted role of intrinsic disorder and multiple LC8 binding events in regulating LC8 transcription. We demonstrate that the long intrinsically disordered C-terminal domain of ASCIZ binds LC8 to form a dynamic ensemble of complexes with a gradient of transcriptional activity that is inversely proportional to LC8 occupancy. The preference …

Life’S Late Digital Revolution And Why It Matters For The Study Of The Origins Of Life, David A. Baum, Niles Lehman

Chemistry Faculty Publications and Presentations

The information contained in life exists in two forms, analog and digital. Analog information is manifest mainly in the differing concentrations of chemicals that get passed from generation to generation and can vary from cell to cell. Digital information is encoded in linear polymers such as DNA and RNA, whose side chains come in discrete chemical forms. Here, we argue that the analog form of information preceded the digital. Acceptance of this dichotomy, and this progression, can help direct future studies on how life originated and initially complexified on the primordial Earth, as well as expected trajectories for other, independent …

Simplified Reversed Chloroquines To Overcome Malaria Resistance To Quinoline-Based Drugs, Bornface Gunsaru, Steven J. Burgess, Westin Morrill, Jane X. Kelly, Shawheen Shomloo, Martin J. Smilkstein, Katherine May Liebman, David H. Peyton

Chemistry Faculty Publications and Presentations

Building on our earlier work of attaching a chemosensitizer (reversal agent) to a known drug pharmacophore, we have now expanded the structure-activity relationship study to include simplified versions of the chemosensitizer. The change from two aromatic rings in this head group to a single ring does not appear to detrimentally affect the antimalarial activity of the compounds. Data from in vitro heme binding and beta-hematin inhibition assays suggest that the single aromatic RCQ compounds retain activities against Plasmodium falciparum similar to those of CQ, although other mechanisms of action may be relevant to their activities.

Topological And Thermodynamic Factors That Influence The Evolution Of Small Networks Of Catalytic Rna Species, Jessica Anne Mellor Yeates, Philippe Nghe, Niles Lehman

Chemistry Faculty Publications and Presentations

An RNA-directed recombination reaction can result in a network of interacting RNA species. It is now becoming increasingly apparent that such networks could have been an important feature of the RNA world during the nascent evolution of life on the Earth. However, the means by which such small RNA networks assimilate other available genotypes in the environment to grow and evolve into the more complex networks that are thought to have existed in the prebiotic milieu are not known. Here, we used the ability of fragments of the Azoarcus group I intron ribozyme to covalently self-assemble via genotype-selfish and genotype-cooperative …

The T6a Modification Acts As A Positive Determinant For The Anticodon Nuclease Prrc, And Is Distinctively Nonessential In Streptococcus Mutans, Jo Marie Bacusmo, Silvia S. Orsini, Jennifer Hu, Michael Demott, Patrick C. Thiaville, Ameer Elfarash, Mellie June Paulines, Diego Rojas-Benítez, Birthe Meineke, Christopher Deutsch, Dirk Iwata-Reuyl, Patrick A. Limbach, Peter C. Dedon, Kelly C. Rice, Stewart Shuman, Valérie De Crécy-Lagard

Chemistry Faculty Publications and Presentations

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALys UUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t⁶A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t⁶A-deficient yeast derivatives, it is shown that t⁶A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t …

The Rna World: 4,000,000,050 Years Old, Niles Lehman

Chemistry Faculty Publications and Presentations

The notion that molecular systems such as RNA can display a wide range of evolutionary processes in the absence of fully formed cellular life continues to gain support. Understanding how RNA can behave in an abiotic context is a key piece of our picture of how life developed and expanded on the Earth, and by proxy, elsewhere. We can study how RNA behaves in this regard through a combination of in vivo work (with small regulatory RNAs and larger catalytic RNAs alike), experimental work in the laboratory, and through powerful analytical and simulation studies. These efforts will not only grant …

Nmr-Based Structural Analysis Of Threonylcarbamoyl-Amp Synthase And Its Substrate Interactions, Kimberly A. Harris, Benjamin G. Bobay, Kathryn L. Sarachan, Alexis F. Sims, Yann Bilbille, Christopher Deutsch, Dirk Iwata-Reuyl, Paul F. Agris

Chemistry Faculty Publications and Presentations

The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in …

Hydrophobic Surfactant Proteins Strongly Induce Negative Curvature, Mariya Chavarha, Ryan W. Loney, Shankar B. Rananavare, Stephen B. Hall

Chemistry Faculty Publications and Presentations

The hydrophobic surfactant proteins SP-B and SP-C greatly accelerate the adsorption of vesicles containing the surfactant lipids to form a film that lowers the surface tension of the air/water interface in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. As with fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, suggesting that adsorption proceeds via a rate-limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. In the studies reported here, we tested whether the surfactant proteins might promote …

Empirical Demonstration Of Environmental Sensing In Catalytic Rna: Evolution Of Interpretive Behavior At The Origins Of Life, Niles Lehman, Tess Bernhard, Brian C. Larson, Andrew J.N. Robinson, Christopher C.B. Southgate

Chemistry Faculty Publications and Presentations

Background: The origins of life on the Earth required chemical entities to interact with their environments in ways that could respond to natural selection. The concept of interpretation, where biotic entities use signs in their environment as proxy for the existence of other items of selective value in their environment, has been proposed on theoretical grounds to be relevant to the origins and early evolution of life. However this concept has not been demonstrated empirically.

Results: Here, we present data that certain catalytic RNA sequences have properties that would enable interpretation of divalent cation levels in their environment. …

Inhalable Constituents Of Thirdhand Tobacco Smoke: Chemical Characterization And Health Impact Considerations, Mohamad Sleiman, Jennifer M. Logue, Wentai Luo, James F. Pankow, Lara A. Gundel, Hugo Destaillats

Chemistry Faculty Publications and Presentations

Tobacco smoke residues lingering in the indoor environment, also termed thirdhand smoke (THS), can be a source of long-term exposure to harmful pollutants. THS composition is affected by chemical transformations and by air–surface partitioning over time scales of minutes to months. This study identified and quantified airborne THS pollutants available for respiratory exposure, identified potential environmental tracers, and estimated health impacts to nonsmokers. In a ventilated 18 m3 laboratory chamber, six cigarettes were machine-smoked, and levels of particulate matter (PM2.5) and 58 volatile organic compounds (VOCs) were monitored during an aging period of 18 h. Results were compared with field …

Candy Flavorings In Tobacco, Jessica E. Brown, Wentai Luo, Lorne M. Isabelle, James F. Pankow

Chemistry Faculty Publications and Presentations

Professor James F. Pankow reveals striking similarities between the patterns in the flavoring chemicals used in flavored tobacco products and those in popular candy and Kool-Aid products. The authors analyzed 12 artificially flavored candy and fruit drink products and compared them to 15 widely-available flavored tobacco products. They found significant overlap in the chemical signatures of the flavor chemicals. Several of the tobacco products contained flavor chemicals at much higher concentrations than in the non-tobacco products.

Differential Effects Of The Hydrophobic Surfactant Proteins On The Formation Of Inverse Bicontinuous Cubic Phases, Mariya Chavarha, Ryan W. Loney, Kamlesh Kumar, Shankar B. Rananavare, Stephen B. Hall

Chemistry Faculty Publications and Presentations

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce formation of the bicontinuous structures and faster adsorption by similar mechanisms, then they should also have differential ability to form the cubic phases. To test this hypothesis, we measured small angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the doserelated …

A Fast Response Highly Selective Probe For The Detection Of Glutathione In Human Blood Plasma, Yixing Guo, Xiaofeng Yang, Lovemore Hakuna, Aabha Barve, Jorge O. Escobedo, Mark Lowry, Robert M. Strongin

Chemistry Faculty Publications and Presentations

A fluorescent probe for glutathione (GSH) detection was developed. Our study indicates a possible mechanism which couples a conjugate addition and micelle-catalyzed large membered ring formation/elimination sequence. This method enables excellent selectivity towards GSH over other biological thiols such as cysteine (Cys) and homocysteine (Hcy). The proposed method is precise with a relative standard deviation (R.S.D) lower than 6% (n = 3) and has been successfully applied to determine GSH in human plasma with recoveries between 99.2% and 102.3%.

Functional Promiscuity Of The Cog0720 Family, Gabriela Phillips, Laura L. Grochowski, Shilah Bonnett, Huimin Xu, Marc Bailly, Crysten Haas-Blaby, Basma El Yacoubi, Dirk Iwata-Reuyl, Robert H. White, Valérie De Crécy-Lagard

Chemistry Faculty Publications and Presentations

The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G⁺) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H₂NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH₄ pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these …

Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments, Bungo Akiyoshi, Krishna K. Sarangapani, Andrew F. Powers, Christian R. Nelson, Steve Reichow, Hugo Arellano-Santoyo, Tamir Gonen, Jeffrey A. Ranish, Charles L. Asbury, Sue Biggins

Chemistry Faculty Publications and Presentations

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be …

Organometallic Iron(Iii)-Salophene Exerts Cytotoxic Properties In Neuroblastoma Cells Via Mapk Activation And Ros Generation, Kyu Kwang Kim, Rakesh K. Singh, Robert M. Strongin, Richard G. Moore, Laurent Brard, Thilo S. Lange

Chemistry Faculty Publications and Presentations

The objective of the present study was to investigate the specific effects of Iron(III)-salophene (Fe-SP) on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB) cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as …

Scoring Function To Predict Solubility Mutagenesis, Ye Tian, Christopher Deutsch, Bala Krishnamoorthy

Chemistry Faculty Publications and Presentations

Background: Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT) protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results: We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to …

Structure Of The Cholera Toxin Secretion Channel In Its Closed State, Steve Reichow, Konstantin V. Korotkov, Wim Gj Hol, Tamir Gonen

Chemistry Faculty Publications and Presentations

The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins including multimeric assemblies like cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the “secretin” GspD. Electron cryomicroscopy reconstruction of the V. cholerae secretin at 19 Å resolution reveals a dodecameric structure reminiscent of a barrel with a large channel at its center that appears to contain a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and binds …

Cooperative Interaction Of Transcription Termination Factors With The Rna Polymerase Ii C-Terminal Domain, Bradley M. Lunde, Steve Reichow, Minkyu Kim, Hyunsuk Suh, Thomas C. Leeper, Fan Yang, Hannes Mutschler, Stephen Buratowski, Anton Meinhart, Gabriele Varani

Chemistry Faculty Publications and Presentations

Phosphorylation of the C-terminal domain of RNA polymerase II controls the co-transcriptional assembly of RNA processing and transcription factors. Recruitment relies on conserved CTDinteracting domains that recognize different CTD phosphoisoforms during the transcription cycle, but the molecular basis for their specificity remains unclear. We show that the CTD-interacting domains of two transcription termination factors, Rtt103 and Pcf11, achieve high affinity and specificity both by specifically recognizing the phosphorylated CTD and by cooperatively binding to neighboring CTD repeats. Single amino acid mutations at the protein-protein interface abolish cooperativity and affect recruitment at the 3′-end processing site in vivo. We suggest that …

Quasispecies-Like Behavior Observed In Catalytic Rna Populations Evolving In A Test Tube, Carolina Diaz Arenas, Niles Lehman

Chemistry Faculty Publications and Presentations

Background: During the RNA World, molecular populations were probably very small and highly susceptible to the force of strong random drift. In conjunction with Muller's Ratchet, this would have imposed difficulties for the preservation of the genetic information and the survival of the populations. Mechanisms that allowed these nascent populations to overcome this problem must have been advantageous.

Results: Using continuous in vitro evolution experimentation with an increased mutation rate imposed by MnCl2, it was found that clonal 100-molecule populations of ribozymes clearly exhibit certain characteristics of a quasispecies. This is the first time this has been seen …

Lipid-Protein Interactions Probed By Electron Crystallography, Steve L. Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid-bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein's architecture where they can affect the function, structure, quaternary assembly and …

Apoptotic And Chemotherapeutic Properties Of Iron (Iii)-Salophene In An Ovarian Cancer Animal Model, Thilo S. Lange, Carolyn Mccourt, Rakesh K. Singh, Kyu Kwang Kim, Ajay P. Singh, Brian S. Luisi, Onur Alptürk, Robert M. Strongin, Laurent Brard

Chemistry Faculty Publications and Presentations

The cytotoxicity of organometallic compounds iron(III)-, cobalt(III)-, manganese(II)-, and copper(II)-salophene (-SP) on platinum-resistant ovarian cancer cell lines was compared. Fe-SP displayed selective cytotoxicity (IC(50) at ~1 muM) against SKOV-3 and OVCAR-3 cell lines while Co-SP caused cytotoxic effects only at higher concentrations (IC(50) at 60 muM) and Cu-SP effects were negligible. High cytotoxicity of Mn-SP (30-60 muM) appeared to be nonspecific because the Mn-chloride salt reduced cell viability similarly. The effect of Fe-SP at 1 muM proved to be ovarian cancer cell selective when compared to a panel of cell lines derived from different tumors. The first irreversible step in …

One Rna Plays Three Roles To Provide Catalytic Activity To A Group I Intron Lacking An Endogenous Internal Guide Sequence, Nilesh Vaidya, Niles Lehman

Chemistry Faculty Publications and Presentations

Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5? end of the molecule, and added back exogenously in trans. The presence of GUG triplets in noncovalent fragments of …

Non-Canonical Binding Of Calmodulin To Aquaporin-0: Implications For Channel Regulation, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homo-tetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show CaM binds the AQP0 C-terminal domain in a calcium dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a non-canonical fashion, suggesting a form of co-operativity between AQP0 monomers. Based on …

Electron Crystallography Of Aquaporins, Simeon Andrews, Steve Reichow, Tamir Gonen

Chemistry Faculty Publications and Presentations

Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

Iron(Iii)-Salophene: An Organometallic Compound With Selective Cytotoxic And Anti-Proliferative Properties In Platinum-Resistant Ovarian Cancer Cells, Thilo S. Lange, Kyu Kwang Kim, Rakesh K. Singh, Robert M. Strongin, Carolyn Mccourt, Laurent Brard

Chemistry Faculty Publications and Presentations

Background: In this pioneer study to the biological activity of organometallic compound Iron(III)-salophene (Fe-SP) the specific effects of Fe-SP on viability, morphology, proliferation, and cell-cycle progression on platinum-resistant ovariancancer cell lines were investigated.

Methodology/Principal Findings: Fe-SP displayed selective cytotoxicity against SKOV-3 and OVCAR-3 (ovarian epithelial adenocarcinoma) cell lines at concentrations between 100 nM and 1 μM, while the viability of HeLa cells (epithelial cervix adenocarcinoma) or primary lung or skin fibroblasts was not affected. SKOV-3 cells in contrast to fibroblasts after treatment with Fe-SP revealed apparent hallmarks of apoptosis including densely stained nuclear granular bodies within fragmented nuclei, highly condensed …

An Embarrassment Of Riches: The Enzymology Of Rna Modification, Dirk Iwata-Reuyl

Chemistry Faculty Publications and Presentations

The maturation of transfer RNA (tRNA) involves extensive chemical modification of the constituent nucleosides and results in the introduction of significant chemical diversity to tRNA. Many of the pathways to these modified nucleosides are characterized by chemically complex transformations, some of which are unprecedented in other areas of biology. To illustrate the scope of the field, recent progress in understanding the enzymology leading to the formation of two distinct classes of modified nucleosides, the thiouridines and queuosine, a 7-deazaguanosine, is reviewed. In particular, recent data validating the involvement of several proposed intermediates in the formation of thiouridines are discussed, including …