Biology Commons^™

Structure Of Mandelate Racemase With Bound Intermediate Analogues Benzohydroxamate And Cupferron, Adam D. Lietzan, Mitesh Nagar, Elisa Pellmann, Jennifer R. Bourque, Stephen L. Bearne, Martin St. Maurice

Biological Sciences Faculty Research and Publications

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg2+-dependent interconversion of the enantiomers of mandelate, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, we determined the X-ray crystal structures of wild-type MR complexed with two analogues of the putative aci-carboxylate intermediate, benzohydroxamate and Cupferron, to 2.2-Å resolution. Benzohydroxamate is shown to be a reasonable mimic of the transition state and/or intermediate because its binding affinity for 21 MR variants correlates well with changes in the free energy of transition …

Inhibitory Effects Of Arabidopsis Earli1 Against Botrytis Cinerea And Bradysia Difformis, Zhen Du, Dan Xu, Lan Li, Yao Shi, Michael Schläppi, Zi-Qin Xu

Biological Sciences Faculty Research and Publications

The aim of this study is to understand the function of EARLI1 in plants subjected to different biotic stresses using EARLI1 overexpressing (OX) and T-DNA knockout (KO) transgenic Arabidopsis lines. Higher levels of expression of EARLI1 in OX lines were confirmed by RT-PCR and Northern blot analysis. The full-length EARLI1 mRNA could not be detected by RT-PCR in KO lines, while only a shorter transcript could be found by RNA gel blotting. In wild-type Col-0 plants (Wt), EARLI1 could be induced by Botrytis cinerea and H₂O₂, indicating this gene might be involved in plant defense system …

La Red Internacional De Inventarios Forestales (Biotree-Net) En Mesoamérica: Avances, Retos Y Perspectivas Futuras, Luis Cayuela, Lucía Gálvez-Bravo, F S. De Albuquerque, Duncan J. Golicher, Mario González-Espinosa, Neptalí Ramírez-Marcial, José M. Rey Benayas, Rakan A. Zahawi, Jorge A. Meave, B. M. Benito, Cristina Garibaldi, I. Chan, Ramón Pérez Pérez, Richard Field, Patricia Balvanera, M. A. Castillo, Blanca L. Figueroa-Rangel, Daniel M. Griffith, Gerald A. Islebe, D. L. Kelly, Miguel Olvera-Vargas, Stefan A. Schnitzer, E. Velazquez, Guadalupe Williams-Linera, Steven W. Brewer, Angélica Camacho-Cruz, Indiana Coronado, Ben De Jong, R. Del Castillo, I. Granzowde La Cerda, Javier Fernández, William Fonseca, Luis Galindo-Jaimes, Thomas W. Gillespie, Benigno González-Rivas, J. E. Gordon, Johanna Hurtado, José Linares, Susan G. Letcher, Scott A. Mangan, V. E. Méndez, Victor Meza, Susana Ochoa-Gaona, C. J. Peterson, Viviana Ruiz-Gutierrez, Kymberley A. Snarr, Fernando Tun Dzul, Mirna Valdez-Hernández, Karin M. Viergever, D. A. White, J. N. Williams, Francisco J. Bonet, Regino Zamora

Biological Sciences Faculty Research and Publications

Conservation efforts in Neotropical regions are often hindered by lack of data, since for many species there is a vacuum of information, and many species have not even been described yet. The International Network of Forest Inventory Plots (BIOTREE-NET) gathers and facilitates access to tree data from forest inventory plots in Mesoamerica, while encouraging data exchange between researchers, managers and conservationists. The information is organised and standardised into a single database that includes spatially explicit data. This article describes the scope and objectives of the network, its progress, and the challenges and future perspectives. The database includes above 50000 tree …

Detection Of Mutant-Huntingtin Aggregation Conformers And Modulation Of Sds-Soluble Fibrillar Oligomers By Small Molecules, Emily M. Sontag, Gregor P. Lotz, Goucheng Yang, Christopher J. Sontag, Brian J. Cummings, Charles G. Glabe, Paul J. Muchowski, Leslie Michels Thompson

Biological Sciences Faculty Research and Publications

The Huntington's disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation …

A Novel Alcohol-Sensitive Site In The M3 Domain Of The Nmda Receptor Glun2a Subunit, Hong Ren, Robert W. Peoples

Biomedical Sciences Faculty Research and Publications

Accumulating studies have demonstrated that the N-methyl-D-aspartate receptor is one of the most important targets of ethanol in the central nervous system. Previous studies from this laboratory have found that one position in the third (F637) and two positions in the fourth (M823 and A825) membrane-associated (M) domains of the N-methyl-D-aspartate receptor GluN2A subunit modulate alcohol action and ion channel gating. Using site-directed mutagenesis and whole-cell patch-clamp recording, we have found an additional position in M3 of the GluN2A subunit, F636, which significantly influences ethanol sensitivity and functionally interacts with F637. Tryptophan substitution at F636 significantly decreased the ethanol IC50, …

A Flagellar A-Kinase Anchoring Protein With Two Amphipathic Helices Forms A Structural Scaffold In The Radial Spoke Complex, Priyanka Sivadas, Jennifer M. Dienes, Martin St. Maurice, William D. Meek, Pinfen Yang

Biological Sciences Faculty Research and Publications

A-kinase anchoring proteins (AKAPs) contain an amphipathic helix (AH) that binds the dimerization and docking (D/D) domain, RIIa, in cAMP-dependent protein kinase A (PKA). Many AKAPs were discovered solely based on the AH–RIIa interaction in vitro. An RIIa or a similar Dpy-30 domain is also present in numerous diverged molecules that are implicated in critical processes as diverse as flagellar beating, membrane trafficking, histone methylation, and stem cell differentiation, yet these molecules remain poorly characterized. Here we demonstrate that an AKAP, RSP3, forms a dimeric structural scaffold in the flagellar radial spoke complex, anchoring through two distinct AHs, the RIIa …

A New Genus Of Miniaturized And Pug-Nosed Gecko From South America (Sphaerodactylidae: Gekkota), Tony Gamble, Juan D. Daza, Guarino R. Colli, Laurie J. Vitt, Aaron M. Bauer

Biological Sciences Faculty Research and Publications

Sphaerodactyl geckos comprise five genera distributed across Central and South America and the Caribbean. We estimated phylogenetic relationships among sphaerodactyl genera using both separate and combined analyses of seven nuclear genes. Relationships among genera were incongruent at different loci and phylogenies were characterized by short, in some cases zero-length, internal branches and poor phylogenetic support at most nodes. We recovered a polyphyletic Coleodactylus, with Coleodactylus amazonicus being deeply divergent from the remaining Coleodactylus species sampled. The C. amazonicus lineage possessed unique codon deletions in the genes PTPN₁₂ and RBMX while the remaining Coleodactylus species had unique codon …

Flexibility In The Patterning And Control Of Axial Locomotor Networks In Lamprey, James T. Buchanan

Biological Sciences Faculty Research and Publications

In lower vertebrates, locomotor burst generators for axial muscles generally produce unitary bursts that alternate between the two sides of the body. In lamprey, a lower vertebrate, locomotor activity in the axial ventral roots of the isolated spinal cord can exhibit flexibility in the timings of bursts to dorsally-located myotomal muscle fibers versus ventrally-located myotomal muscle fibers. These episodes of decreased synchrony can occur spontaneously, especially in the rostral spinal cord where the propagating body waves of swimming originate. Application of serotonin, an endogenous spinal neurotransmitter known to presynaptically inhibit excitatory synapses in lamprey, can promote decreased synchrony of dorsal–ventral …

Identifying Polyglutamine Protein Species In Situ That Best Predict Neurodegeneration, Jason Miller, Montserrat Arrasate, Elizabeth Brooks, Clare Peter Libeu, Justin Legleiter, Danny Hatters, Jessica Curtis, Kenneth Cheung, Preethi Krishnan, Siddhartha Mitra, Kartika Widjaja, Benjamin A. Shaby, Gregor P. Lotz, Yvonne Newhouse, Emily M. Sontag, Alex Osmand, Michelle Gray, Vanitha Thulasiramin, Frederic Saudou, Mark Segal, X William Yang, Eliezer Masliah, Leslie M. Thompson, Paul J. Muchowski, Karl H. Weisgraber, Steven Finkbeiner

Biological Sciences Faculty Research and Publications

Polyglutamine (polyQ) stretches exceeding a threshold length confer a toxic function to proteins that contain them and cause at least nine neurological disorders. The basis for this toxicity threshold is unclear. Although polyQ expansions render proteins prone to aggregate into inclusion bodies, this may be a neuronal coping response to more toxic forms of polyQ. The exact structure of these more toxic forms is unknown. Here we show that the monoclonal antibody 3B5H10 recognizes a species of polyQ protein in situ that strongly predicts neuronal death. The epitope selectively appears among some of the many low-molecular-weight conformational states assumed by …

Minimizing Bias In Biomass Allometry: Model Selection And Log‐Transformation Of Data, Joseph Mascaro, Creighton M. Litton, R. Flint Hughes, Amanda Uowolo, Stefan A. Schnitzer

Biological Sciences Faculty Research and Publications

Nonlinear regression is increasingly used to develop allometric equations for forest biomass estimation (i.e., as opposed to the traditional approach of log‐transformation followed by linear regression). Most statistical software packages, however, assume additive errors by default, violating a key assumption of allometric theory and possibly producing spurious models. Here, we show that such models may bias stand‐level biomass estimates by up to 100 percent in young forests, and we present an alternative nonlinear fitting approach that conforms with allometric theory.

Spinal Locomotor Inputs To Individually Identified Reticulospinal Neurons In The Lamprey, James T. Buchanan

Biological Sciences Faculty Research and Publications

Locomotor feedback signals from the spinal cord to descending brain stem neurons were examined in the lamprey using the uniquely identifiable reticulospinal neurons, the Müller and Mauthner cells. The same identified reticulospinal neurons were recorded in several preparations, under reduced conditions, to address whether an identified reticulospinal neuron shows similar locomotor-related oscillation timing from animal to animal and whether these timing signals can differ significantly from other identified reticulospinal neurons. Intracellular recordings of membrane potential in identified neurons were made in an isolated brain stem-spinal cord preparation with a high-divalent cation solution on the brain stem to suppress indirect neural …

The Pleiotropic Effects Of Lc8 On The Anchoring Protein In Flagellar Radial Spoke Complex, Anjali Gupta

Dissertations (1934 -)

The goal of this thesis is to elucidate the assembly mechanism of macromolecular complexes in the microtubule-based axoneme in flagella. The axoneme is comprised of more than 400 distinct polypeptides. While all axonemal proteins are synthesized in the cell body, they need to be imported to the tip of the growing flagella for the final assembly. They are then assembled into distinctive structural complexes and integrated into precise positions relative to each other. The assembled macromolecular complexes operate in concert enabling the axoneme to beat rhythmically as a biological machine. This arduous process involves multiple reactions and meticulous regulation. However, …

Mechanisms Of Neuronal Death Induced By Environmental Toxicants In Murine Cortical Culture, Travis Rush

Dissertations (1934 -)

This study was directed at examining the neurotoxic mechanisms of several classes of environmental toxicants implicated in neurodegenerative disease. Primary cortical cultures were exposed to organophosphorus pesticides, heavy metals and the cyanobacterial toxin, beta-N-methylamino-L-alanine (BMAA). Several components relating to neuronal injury were assessed in each study and novel aspects are described.

The main action of organophosphorous insecticides is generally believed to be the inhibition of acetylcholinesterase. However, these compounds are now recognized to inhibit many other enzymes and cause neuronal death through a variety of mechanisms. I found that exposure to chlorpyrifos or diazinon caused concentration-dependent neurotoxicity that could not …

A Tight Coupling Between Β2Y97 And Β2F200 Of The GabaA Receptor Mediates Gaba Binding, Phu N. Tran, Kurt T. Laha, David A. Wagner

Biological Sciences Faculty Research and Publications

The GABA_A receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α₁R67, α₁R120, α₁R132, and β₂R207) and two aromatic residues (β₂Y97 and β₂F200) …

Mouse Versus Rat: Profound Differences In Meiotic Regulation At The Level Of The Isolated Oocyte, Stephen M. Downs

Biological Sciences Faculty Research and Publications

Cumulus cell-enclosed oocytes (CEO), denuded oocytes (DO), or dissected follicles were obtained 44–48 hr after priming immature mice (20–23 days old) with 5 IU or immature rats (25–27 days old) with 12.5 IU of equine chorionic gonadotropin, and exposed to a variety of culture conditions. Mouse oocytes were more effectively maintained in meiotic arrest by hypoxanthine, dbcAMP, IBMX, milrinone, and 8-Br-cGMP. Atrial natriuretic peptide, a guanylate cyclase activator, suppressed maturation in CEO from both species, but mycophenolic acid reversed IBMX-maintained meiotic arrest in mouse CEO with little activity in rat CEO. IBMX-arrested mouse, but not rat, CEO were induced to …

The Discovery And Exploration Of A Universal Targeting Mechanism In Eukaryotic Cells, Priyanka Sivadas

Dissertations (1934 -)

A wide range of eukaryotic organisms generate motile cilia and flagella. These slender organelles beat rhythmically to move the surrounding fluid or to propel cells in aqueous environment. Organisms use these powerful yet nimble organelles to forage, evade, adapt and mate. The machinery that drives this tightly controlled movement is the sophisticated microtubule-based axoneme. As it is critical for the survival of individual species, this machinery has largely been preserved to the molecular level throughout evolution. Proteomic studies have shown that most proteins in this biological machine consist of molecular modules commonly used in the cell body. But the usage …

Rhythmic Leptin Is Required For Weight Gain From Circadian Desynchronized Feeding In The Mouse, Deanna M. Arble, Martha H. Vitaterna, Fred W. Turek

Biological Sciences Faculty Research and Publications

The neuroendocrine and metabolic effects of leptin have been extensively researched since the discovery, and the later identification, of the leptin gene mutated within the ob/ob mouse. Leptin is required for optimal health in a number of physiological systems (e.g. fertility, bone density, body weight regulation). Despite the extensive leptin literature and many observations of leptin’s cyclical pattern over the 24-hour day, few studies have specifically examined how the circadian rhythm of leptin may be essential to leptin signaling and health. Here we present data indicating that a rhythmic leptin profile (e.g. 1 peak every 24 hours) leads to excessive …

Uncovering New Functions For Micrornas In Caenorhabditis Elegans, Allison L. Abbott

Biological Sciences Faculty Research and Publications

In the race to understand microRNA (miRNA) functions in development and physiology, Caenorhabditis elegans investigators were the first out of the gate with the cloning and analysis of the lin-4 and let-7 miRNAs [1,2]. The starting point of strong, penetrant loss of function phenotypes facilitated these advancements. However, subsequent functional analysis of miRNAs in C. elegans was hampered by the lack of easily observable loss-of-function phenotypes [3]. There are several possible models to account for this observation. First, redundancy between related miRNAs can account for the absence of phenotypes in mutants missing individual miRNA genes [4,5]. Second, miRNAs may also …

Truncation Of The Mrp20 Protein Reveals New Ribosome‐Assembly Subcomplex In Mitochondria, Jasvinder Kaur, Rosemary A. Stuart

Biological Sciences Faculty Research and Publications

Mitochondrial ribosomal protein 20 (Mrp20) is a component of the yeast mitochondrial large (54S) ribosomal subunit and is homologous to the bacterial L23 protein, located at the ribosomal tunnel exit site. The carboxy‐terminal mitochondrial‐specific domain of Mrp20 was found to have a crucial role in the assembly of the ribosomes. A new, membrane‐bound, ribosomal‐assembly subcomplex composed of known tunnel‐exit‐site proteins, an uncharacterized ribosomal protein, MrpL25, and the mitochondrial peroxiredoxin (Prx), Prx1, accumulates in an mrp20ΔC yeast mutant. Finally, data supporting the idea that the inner mitochondrial membrane acts as a platform for the ribosome assembly process are discussed.

Influence Of Earli1‐Like Genes On Flowering Time And Lignin Synthesis Of Arabidopsis Thaliana, Y. Shi, X. Zhang, Z.-Y. Xu, C. Zhang, Michael Schläppi, Z.-Q. Xu

Biological Sciences Faculty Research and Publications

EARLI1 encodes a 14.7 kDa protein in the cell wall, is a member of the PRP (proline‐rich protein) family and has multiple functions, including resistance to low temperature and fungal infection. RNA gel blot analyses in the present work indicated that expression of EARLI1‐like genes, EARLI1, At4G12470 and At4G12490, was down‐regulated in Col‐FRI‐Sf2 RNAi plants derived from transformation with Agrobacteriumstrain ABI, which contains a construct encoding a double‐strand RNA targeting 8CM of EARLI1. Phenotype analyses revealed that Col‐FRI‐Sf2 RNAi plants of EARLI1 flowered earlier than Col‐FRI‐Sf2 wild‐type plants. The average …

Self-Assembly Of Escherichia Coli Mutl And Its Complexes With Dna, Anita Niedziela-Majka, Nasib K. Maluf, Edwin Antony, Timothy M. Lohman

Biological Sciences Faculty Research and Publications

The Escherichia coli MutL protein regulates the activity of several enzymes, including MutS, MutH, and UvrD, during methyl-directed mismatch repair of DNA. We have investigated the self-association properties of MutL and its binding to DNA using analytical sedimentation velocity and equilibrium. Self-association of MutL is quite sensitive to solution conditions. At 25 °C in Tris at pH 8.3, MutL assembles into a heterogeneous mixture of large multimers. In the presence of potassium phosphate at pH 7.4, MutL forms primarily stable dimers, with the higher-order assembly states suppressed. The weight-average sedimentation coefficient of the MutL dimer in this buffer (s̅ …

New Insights On Sarcoplasmic Reticulum Calcium Regulation In Muscle Fatigue, Robert H. Fitts

Biological Sciences Faculty Research and Publications

A consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca²⁺ transient, which is thought to result primarily from a reduced Ca²⁺ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) (Fig. 1). This in turn is thought to contribute to the loss in muscle force and power (2). In the past 20 years, the important proteins at the t-tubule SR junction have been identified (Fig. 1), and considerable progress has been made in understanding the molecular mechanism by which t-tubular charge induces SR Ca²⁺ release. However, the …

Soil Microbes Regulate Ecosystem Productivity And Maintain Species Diversity, Stefan A. Schnitzer, John Klironomos

Biological Sciences Faculty Research and Publications

One of the major goals in ecology is to determine the mechanisms that drive the asymptotic increase in ecosystem productivity with plant species diversity. Niche complementarity, the current paradigm for the asymptotic diversity-productivity pattern, posits that the addition of species to a community increases productivity because each species specializes on different resources and thus can more thoroughly utilize the available resources. At higher diversity the increase in productivity decreases because resources become limiting, resulting in the classic asymptotic diversity-productivity pattern. An alternative but less tested explanation is that density-dependent disease from species-specific soil microbes drive the diversity-productivity relationship by increasing …

A Rice Plastidial Nucleotide Sugar Epimerase Is Involved In Galactolipid Biosynthesis And Improves Photosynthetic Efficiency, Chunlai Li, Yiqin Wang, Linchuan Liu, Yingchun Hu, Fengxia Zhang, Sod Mergen, Guodong Wang, Michael Schläppi, Chengcai Chu

Biological Sciences Faculty Research and Publications

Photosynthesis is the final determinator for crop yield. To gain insight into genes controlling photosynthetic capacity, we selected from our large T-DNA mutant population a rice stunted growth mutant with decreased carbon assimilate and yield production named photoassimilate defective1 (phd1). Molecular and biochemical analyses revealed that PHD1 encodes a novel chloroplast-localized UDP-glucose epimerase (UGE), which is conserved in the plant kingdom. The chloroplast localization of PHD1 was confirmed by immunoblots, immunocytochemistry, and UGE activity in isolated chloroplasts, which was approximately 50% lower in the phd1-1 mutant than in the wild type. In addition, the amounts of …

Sequential Assembly Of Flagellar Radial Spokes, Dennis R. Diener, Pinfen Yang, Stefan Geimer, Douglas G. Cole, Winfield S. Sale, Joel L. Rosenbaum

Biological Sciences Faculty Research and Publications

The unicellular alga Chlamydomonas can assemble two 10 μm flagella in 1 h from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella. On the axoneme, radial spokes are “T” shaped structures with a head of five proteins and a stalk of 18 proteins that sediment together at 20S. In the cell body, radial spokes are partially assembled; about half of the radial spoke proteins (RSPs) form …

Mathematical Modeling And Dynamical Analysis Of The Operation Of The Hypothalamus - Pituitary - Thyroid (Hpt) Axis In Autoimmune (Hashimoto's) Thyroiditis, Balamurugan Pandiyan

Dissertations (1934 -)

This thesis is a mathematical modeling study of the operation of the negative feedback control through the hypothalamus-pituitary- thyroid (HPT) axis in autoimmune (Hashimoto's) thyroiditis. Negative feedback control through the HPT axis is a mechanism in which the high levels of thyroid hormone; free thyroxine (FT4) in the blood inhibits the secretion of the pituitary hormone, thyroid stimulating hormone (TSH) into the blood. Similarly, the low levels of free thyroxine (FT4) sensed by the pituitary gland and then it secretes thyroid stimulating hormone (TSH) into the blood. Autoimmune (Hashimoto's) thyroiditis is a disease in which the immune system turns against …

Relating Methanogen Community Structure To Function In Anaerobic Wastewater Digesters, Rachel Morris

Dissertations (1934 -)

Anaerobic wastewater treatment is an environmentally and economically beneficial biotechnology in which the degradation of organic compounds in industrial and municipal wastewaters results in the production of methane, an alternative energy source. The degradation of organic waste is carried out by an interdependent microbial community; and the Archaea known as the methanogens complete one of the final steps, producing methane. However, the contribution of methanogens and the community ecology of anaerobic digesters are just beginning to be understood. Specifically, links between methanogen community structure and the successful transformation of organic pollutants to methane have not been clearly defined. In order …

Unexpected Accumulation Of Ncm5U And Ncm5S2U In A Trm9 Mutant Suggests An Additional Step In The Synthesis Of Mcm5U And Mcm5S2U, Changchun Chen, Bo Huang, James T. Anderson, Anders S. Byström

Biological Sciences Faculty Research and Publications

Background

Transfer RNAs are synthesized as a primary transcript that is processed to produce a mature tRNA. As part of the maturation process, a subset of the nucleosides are modified. Modifications in the anticodon region often modulate the decoding ability of the tRNA. At position 34, the majority of yeast cytosolic tRNA species that have a uridine are modified to 5-carbamoylmethyluridine (ncm⁵U), 5-carbamoylmethyl-2′-O-methyluridine (ncm⁵Um), 5-methoxycarbonylmethyl-uridine (mcm⁵U) or 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U). The formation of mcm⁵ and ncm⁵ side chains involves a complex pathway, where the last step in formation of …

The Rna Helicase Mtr4p Modulates Polyadenylation In The Tramp Complex, Huijue Jia, Xuying Wang, Fei Liu, Ulf-Peter Guenther, Sukanya Srinivasan, James T. Anderson, Eckhard Jankowsky

Biological Sciences Faculty Research and Publications

Many steps in nuclear RNA processing, surveillance, and degradation require TRAMP, a complex containing the poly(A) polymerase Trf4p, the Zn-knuckle protein Air2p, and the RNA helicase Mtr4p. TRAMP polyadenylates RNAs designated for decay or trimming by the nuclear exosome. It has been unclear how polyadenylation by TRAMP differs from polyadenylation by conventional poly(A) polymerase, which produces poly(A) tails that stabilize RNAs. Using reconstituted S. cerevisiae TRAMP, we show that TRAMP inherently suppresses poly(A) addition after only 3–4 adenosines. This poly(A) tail length restriction is controlled by Mtr4p. The helicase detects the number of 3′-terminal adenosines and, over several adenylation steps, …

Probing The Allosteric Activation Of Pyruvate Carboxylase Using 2′,3′-O-(2,4,6-Trinitrophenyl) Adenosine 5′-Triphosphate As A Fluorescent Mimic Of The Allosteric Activator Acetyl Coa, Abdussalam Adina-Zada, Rasmani Hazra, Chutima Sereerukb, Sarawut Jitrapakdee, Tonya N. Zeczycki, Martin St. Maurice, W Wallace Cleland, John C. Wallace, Paul V. Attwood

Biological Sciences Faculty Research and Publications

2′,3′-O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of …