FUNCTIONAL and MECHANISTIC CHARACTERIZATION of TWO TRNA MODIFYING ENZYMES LAURA CAROLE KEFFER-WILKES Master of Science, Universi
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Us 2018 / 0353618 A1
US 20180353618A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0353618 A1 Burkhardt et al. (43 ) Pub . Date : Dec . 13 , 2018 (54 ) HETEROLOGOUS UTR SEQUENCES FOR Publication Classification ENHANCED MRNA EXPRESSION (51 ) Int. Cl. A61K 48 / 00 ( 2006 .01 ) (71 ) Applicant : Moderna TX , Inc ., Cambridge , MA C12N 15 / 10 (2006 .01 ) (US ) C12N 15 /67 ( 2006 .01 ) (72 ) Inventors : David H . Burkhardt , Somerville , MA A61K 31 / 7115 ( 2006 .01 ) (US ) ; Romesh R . Subramanian , A61K 31 /712 ( 2006 .01 ) A61K 31 / 7125 (2006 . 01 ) Framingham , MA (US ) ; Christian A61P 1 / 16 ( 2006 .01 ) Cobaugh , Newton Highlands , MA (US ) A61P 31/ 14 (2006 .01 ) (52 ) U . S . CI. (73 ) Assignee : Moderna TX , Inc. , Cambridge , MA CPC . .. A61K 48 /0058 ( 2013 .01 ) ; C12N 15 / 1086 (US ) ( 2013 . 01 ) ; A61K 48 / 0066 ( 2013 .01 ) ; C12N 15 /67 ( 2013 .01 ) ; A61P 31 / 14 ( 2018 . 01 ) ; A61K ( 21 ) Appl . No . : 15 / 781, 827 31/ 712 (2013 .01 ) ; A61K 31/ 7125 ( 2013 .01 ) ; A61P 1 / 16 ( 2018 .01 ) ; A61K 31/ 7115 ( 22 ) PCT Filed : Dec . 9 , 2016 ( 2013 .01 ) ( 86 ) PCT No. : PCT/ US2016 / 065796 (57 ) ABSTRACT $ 371 ( c ) ( 1 ) , mRNAs containing an exogenous open reading frame (ORF ) ( 2 ) Date : flanked by a 5 ' untranslated region (UTR ) and a 3 ' UTR is Jun . 6 , 2018 provided , wherein the 5 ' and 3 ' UTRs are derived from a naturally abundant mRNA in a tissue . Also provided are Related U . S . Application Data methods for identifying the 5 ' and 3 ' UTRs, and methods for (60 ) Provisional application No . 62 / 265 , 233 , filed on Dec. making and using the mRNAs . -
Expanding the Toolkit of Protein Engineering: Towards Multiple Simultaneous in Vivo Incorporation of Noncanonical Amino Acids
TECHNISCHE UNIVERSITÄT MÜNCHEN Max-Planck-Institut für Biochemie Expanding the Toolkit of Protein Engineering: Towards Multiple Simultaneous In Vivo Incorporation of Noncanonical Amino Acids Michael G. Hösl Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. Michael Groll Prüfer der Dissertation: 1. Univ.-Prof. Dr. Nediljko Budisa, Technische Universität Berlin 2. Univ.-Prof. Dr. Thomas Kiefhaber 3. Univ.-Prof. Dr. Johannes Buchner Die Dissertation wurde am 01.02.11 bei der Technischen Universität München eingereicht und durch die Fakultät für Chemie am 03.03.11 angenommen. To Teresa Ariadna García-Grajalva Lucas who influenced the idea of TAG → AGG switch just by the existence of her name Sleeping is giving in, no matter what the time is. Sleeping is giving in, so lift those heavy eyelids. People say that you'll die faster than without water. But we know it's just a lie, scare your son, scare your daughter. People say that your dreams are the only things that save ya. Come on baby in our dreams, we can live on misbehavior. The Arcade Fire Parts of this work were published as listed below: Hoesl, MG, Budisa, N. Expanding and engineering the genetic code in a single expression experiment. ChemBioChem 2011, 12, 552-555. Further publications: Hoesl, MG*, Staudt, H*, Dreuw, A, Budisa, N, Grininger, M, Oesterhelt, D, Wachtveitl, J. Manipulating the eletron transfer in Dodecin by isostructual noncanonical Trp analogs. 2011, [in preparation]. *authors contributed equally to this work Nehring, S*, Hoesl, MG*, Acevedo-Rocha, CG*, Royter, M, Wolschner, C, Wiltschi, B, Budisa, N, Antranikian, G. -
Broad and Adaptable RNA Structure Recognition by the Human Interferon-Induced Tetratricopeptide Repeat Protein IFIT5
Broad and adaptable RNA structure recognition by the human interferon-induced tetratricopeptide repeat protein IFIT5 George E. Katibaha, Yidan Qinb, David J. Sidoteb, Jun Yaob, Alan M. Lambowitzb,1, and Kathleen Collinsa,1 aDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and bInstitute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712 Contributed by Alan M. Lambowitz, July 8, 2014 (sent for review April 29, 2014; reviewed by Sandra L. Wolin, Eric Phizicky, and Michael Gale, Jr.) Interferon (IFN) responses play key roles in cellular defense against copy numbers and combinations of four distinct IFIT proteins pathogens. Highly expressed IFN-induced proteins with tetratrico- (IFIT1, 2, 3, and 5) even within mammals, generated by paralog peptide repeats (IFITs) are proposed to function as RNA binding expansions and/or gene deletions, including the loss of IFIT5 in proteins, but the RNA binding and discrimination specificities of IFIT mice and rats (14). Human IFIT1, IFIT2, and IFIT3 coassemble proteins remain unclear. Here we show that human IFIT5 has in cells into poorly characterized multimeric complexes that ex- comparable affinity for RNAs with diverse phosphate-containing 5′- clude IFIT5 (15, 16). Recombinant IFIT family proteins range ends, excluding the higher eukaryotic mRNA cap. Systematic muta- from monomer to multimer, with crystal structures solved for genesis revealed that sequence substitutions in IFIT5 can alternatively a human IFIT2 homodimer (17), the human IFIT5 monomer expand or introduce bias in protein binding to RNAs with 5′ mono- (16, 18, 19), and an N-terminal fragment of human IFIT1 (18). -
Genome-Wide Investigation of Cellular Functions for Trna Nucleus
Genome-wide Investigation of Cellular Functions for tRNA Nucleus- Cytoplasm Trafficking in the Yeast Saccharomyces cerevisiae DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Hui-Yi Chu Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2012 Dissertation Committee: Anita K. Hopper, Advisor Stephen Osmani Kurt Fredrick Jane Jackman Copyright by Hui-Yi Chu 2012 Abstract In eukaryotic cells tRNAs are transcribed in the nucleus and exported to the cytoplasm for their essential role in protein synthesis. This export event was thought to be unidirectional. Surprisingly, several lines of evidence showed that mature cytoplasmic tRNAs shuttle between nucleus and cytoplasm and their distribution is nutrient-dependent. This newly discovered tRNA retrograde process is conserved from yeast to vertebrates. Although how exactly the tRNA nuclear-cytoplasmic trafficking is regulated is still under investigation, previous studies identified several transporters involved in tRNA subcellular dynamics. At least three members of the β-importin family function in tRNA nuclear-cytoplasmic intracellular movement: (1) Los1 functions in both the tRNA primary export and re-export processes; (2) Mtr10, directly or indirectly, is responsible for the constitutive retrograde import of cytoplasmic tRNA to the nucleus; (3) Msn5 functions solely in the re-export process. In this thesis I focus on the physiological role(s) of the tRNA nuclear retrograde pathway. One possibility is that nuclear accumulation of cytoplasmic tRNA serves to modulate translation of particular transcripts. To test this hypothesis, I compared expression profiles from non-translating mRNAs and polyribosome-bound translating mRNAs collected from msn5Δ and mtr10Δ mutants and wild-type cells, in fed or acute amino acid starvation conditions. -
A Protein Subunit of Human Rnase P, Rpp14, and Its Interacting Partner, OIP2, Have 335 Exoribonuclease Activity
A protein subunit of human RNase P, Rpp14, and its interacting partner, OIP2, have 335 exoribonuclease activity Taijiao Jiang and Sidney Altman* Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520 Contributed by Sidney Altman, February 12, 2002 -The processing of precursor tRNAs at their 5 and 3 termini is a presence of [␣-32P]UTP. Internally labeled SupS1-3Ј was pre fundamental event in the biosynthesis of tRNA. RNase P is gener- pared from internally labeled 5Ј-SupS1-3Ј by removing the 5Ј ally responsible for endonucleolytic removal of a leader sequence leader sequence by using purified human RNase P. For the of precursor tRNA to generate the mature 5 terminus. However, synthesis of 5Ј-32P-labeled 5Ј-Tyr-3Ј, transcription reactions were much less is known about the RNase P counterparts or other performed with nonlabeled NTPs, the RNA was purified and proteins that are active at the tRNA 3 terminus. Here we show that then was treated with calf intestinal phosphatase, extracted two one of the human RNase P subunits, Rpp14, together with one of times with phenol͞chloroform and one time with chloroform. its interacting protein partners, OIP2, is a 335 exoribonuclease Recovery was by ethanol precipitation. 5Ј-Tyr-3Ј was labeled .with a phosphorolytic activity that processes the 3 terminus of with [␥-32P]ATP by using polynucleotide kinase precursor tRNA. Immunoprecipitates of a crude human RNase P Adult enhancer factor-1 (AEF) RNA, a single-stranded RNA complex can process both ends of precursor tRNA by hydrolysis, from Drosophila melanogaster (12), was labeled as indicated by but purified RNase P has no exonuclease activity. -
B Number Gene Name Strand Orientation Protein Length Mrna
list list sample) short list predicted B number Operon ID Gene name assignment Protein length mRNA present mRNA intensity Gene description Protein detected - Strand orientation Membrane protein detected (total list) detected (long list) membrane sample Proteins detected - detected (short list) # of tryptic peptides # of tryptic peptides # of tryptic peptides # of tryptic peptides # of tryptic peptides Functional category detected (membrane Protein detected - total Protein detected - long b0001 thrL + 21 1344 P 1 0 0 0 0 thr operon leader peptide Metabolism of small molecules 1 b0002 thrA + 820 13624 P 39 P 18 P 18 P 18 P(m) 2 aspartokinase I, homoserine dehydrogenase I Metabolism of small molecules 1 b0003 thrB + 310 6781 P 9 P 3 3 P 3 0 homoserine kinase Metabolism of small molecules 1 b0004 thrC + 428 15039 P 18 P 10 P 11 P 10 0 threonine synthase Metabolism of small molecules 1 b0005 b0005 + 98 432 A 5 0 0 0 0 orf, hypothetical protein Open reading frames 2 b0006 yaaA - 258 1047 P 11 P 1 2 P 1 0 orf, hypothetical protein Open reading frames 3 b0007 yaaJ - 476 342 P 8 0 0 0 0 MP-GenProt-PHD inner membrane transport protein Miscellaneous 4 b0008 talB + 317 20561 P 20 P 13 P 16 P 13 0 transaldolase B Metabolism of small molecules 5 b0009 mog + 195 1296 P 7 0 0 0 0 required for the efficient incorporation of molybdate into molybdoproteins Metabolism of small molecules 6 b0010 yaaH - 188 407 A 2 0 0 0 0 PHD orf, hypothetical protein Open reading frames 7 b0011 b0011 - 237 338 P 13 0 0 0 0 putative oxidoreductase Miscellaneous 8 b0012 htgA -
A Possible Functional Link Between RNA Degradation and Transcription in Bacillus Subtilis
A possible functional link between RNA degradation and transcription in Bacillus subtilis Dissertation for the award of the degree “Doctor of Philosophy” Division of Mathematics and Natural Sciences of the Georg-August-University Göttingen within the program “Microbiology and Biochemistry” of the Georg-August University School of Science (GAUSS) submitted by Martin Benda from Prague (Czech Republic) Göttingen 2020 Thesis Committee Prof. Dr. Jörg Stülke (Supervisor and 1st Reviewer) Institute for Microbiology and Genetics, Department of General Microbiology, Georg-August-University Göttingen Prof. Dr. Rolf Daniel (2nd Reviewer) Institute for Microbiology and Genetics, Department of Genomic and Applied Microbiology, Georg-August- University Göttingen Prof. Dr. Fabian M. Commichau Institute for Biotechnology, Department of Synthetic Microbiology, Brandenburg University of Technology Cottbus-Senftenberg Additional members of the Examination Board Prof. Dr. Markus Bohnsack Department of Molecular Biology, University Medical Center Göttingen Prof. Dr. Patrick Cramer Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen Prof. Dr. Stefanie Pöggeler Institute for Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg- August-University Göttingen Date of oral examination: September 17th, 2020 I hereby declare that this Ph.D. thesis entitled “A possible functional link between RNA degradation and transcription in Bacillus subtilis” has been written independently and with no other sources and aids than quoted. Martin Benda Acknowledgements First of all, I would like to express my deep thanks to my supervisor Prof. Dr. Jörg Stülke for the great guidance during this work. He put his full trust in me and gave me the opportunity to work on this intricate, but engaging topic of RNA degradation. -
An Investigation of Bacterial Ribonucleases As an Antibiotic Target Ashley Denise Frazier East Tennessee State University
East Tennessee State University Digital Commons @ East Tennessee State University Electronic Theses and Dissertations Student Works 5-2012 An Investigation of Bacterial Ribonucleases as an Antibiotic Target Ashley Denise Frazier East Tennessee State University Follow this and additional works at: https://dc.etsu.edu/etd Part of the Bacteriology Commons Recommended Citation Frazier, Ashley Denise, "An Investigation of Bacterial Ribonucleases as an Antibiotic Target" (2012). Electronic Theses and Dissertations. Paper 1417. https://dc.etsu.edu/etd/1417 This Dissertation - Open Access is brought to you for free and open access by the Student Works at Digital Commons @ East Tennessee State University. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Digital Commons @ East Tennessee State University. For more information, please contact [email protected]. An Investigation of Bacterial Ribonucleases as an Antibiotic Target A dissertation presented to the faculty of the Department of Biochemistry and Molecular Biology East Tennessee State University In partial fulfillment of the requirements for the degree Doctor of Philosophy of Biomedical Sciences by Ashley Denise Frazier May 2012 W. Scott Champney, Ph.D., Chair Mitchell E. Robinson, Ph.D. Douglas P. Thewke, Ph.D. J. Russell Hayman, Ph.D. Bert C. Lampson, Ph.D. Keywords: Ribonuclease Mutants, Ribosomal Subunit Assembly, Aminoglycosides, Escherichia coli, Staphylococcus aureus, Protein Synthesis, Vanadyl Ribonucleoside Complex ABSTRACT An Investigation of Bacterial Ribonucleases as an Antibiotic Target by Ashley Denise Frazier Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance. -
Supplemental Table S1: Comparison of the Deleted Genes in the Genome-Reduced Strains
Supplemental Table S1: Comparison of the deleted genes in the genome-reduced strains Legend 1 Locus tag according to the reference genome sequence of B. subtilis 168 (NC_000964) Genes highlighted in blue have been deleted from the respective strains Genes highlighted in green have been inserted into the indicated strain, they are present in all following strains Regions highlighted in red could not be deleted as a unit Regions highlighted in orange were not deleted in the genome-reduced strains since their deletion resulted in severe growth defects Gene BSU_number 1 Function ∆6 IIG-Bs27-47-24 PG10 PS38 dnaA BSU00010 replication initiation protein dnaN BSU00020 DNA polymerase III (beta subunit), beta clamp yaaA BSU00030 unknown recF BSU00040 repair, recombination remB BSU00050 involved in the activation of biofilm matrix biosynthetic operons gyrB BSU00060 DNA-Gyrase (subunit B) gyrA BSU00070 DNA-Gyrase (subunit A) rrnO-16S- trnO-Ala- trnO-Ile- rrnO-23S- rrnO-5S yaaC BSU00080 unknown guaB BSU00090 IMP dehydrogenase dacA BSU00100 penicillin-binding protein 5*, D-alanyl-D-alanine carboxypeptidase pdxS BSU00110 pyridoxal-5'-phosphate synthase (synthase domain) pdxT BSU00120 pyridoxal-5'-phosphate synthase (glutaminase domain) serS BSU00130 seryl-tRNA-synthetase trnSL-Ser1 dck BSU00140 deoxyadenosin/deoxycytidine kinase dgk BSU00150 deoxyguanosine kinase yaaH BSU00160 general stress protein, survival of ethanol stress, SafA-dependent spore coat yaaI BSU00170 general stress protein, similar to isochorismatase yaaJ BSU00180 tRNA specific adenosine -
From Trna Modifications to DNA Adducts and Back to Miscoding Ribonucleotides F
Guengerich and Ghodke Genes and Environment (2021) 43:24 https://doi.org/10.1186/s41021-021-00199-x REVIEW Open Access Etheno adducts: from tRNA modifications to DNA adducts and back to miscoding ribonucleotides F. Peter Guengerich* and Pratibha P. Ghodke Abstract Etheno (and ethano) derivatives of nucleic acid bases have an extra 5-membered ring attached. These were first noted as wyosine bases in tRNAs. Some were fluorescent, and the development of etheno derivatives of adenosine, cytosine, and guanosine led to the synthesis of fluorescent analogs of ATP, NAD+, and other cofactors for use in biochemical studies. Early studies with the carcinogen vinyl chloride revealed that these modified bases were being formed in DNA and RNA and might be responsible for mutations and cancer. The etheno bases are also derived from other carcinogenic vinyl monomers. Further work showed that endogenous etheno DNA adducts were present in animals and humans and are derived from lipid peroxidation. The chemical mechanisms of etheno adduct formation involve reactions with bis-electrophiles generated by cytochrome P450 enzymes or lipid peroxidation, which have been established in isotopic labeling studies. The mechanisms by which etheno DNA adducts miscode have been studied with several DNA polymerases, aided by the X-ray crystal structures of these polymerases in mispairing situations and in extension beyond mispairs. Repair of etheno DNA adduct damage is done primarily by glycosylases and also by the direct action of dioxygenases. Some human DNA polymerases (η, κ) can insert bases opposite etheno adducts in DNA and RNA, and the reverse transcriptase activity may be of relevance with the RNA etheno adducts. -
The Evolution and Functions of Trna Modifications
life Review From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications Katherine M. McKenney 1,2 and Juan D. Alfonzo 1,2,3,* 1 The Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA; [email protected] 2 The Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA 3 Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA * Correspondence: [email protected]; Tel.: +1-614-292-0004 Academic Editors: Adrian Gabriel Torres and David Deamer Received: 11 January 2016; Accepted: 7 March 2016; Published: 14 March 2016 Abstract: All nucleic acids in cells are subject to post-transcriptional chemical modifications. These are catalyzed by a myriad of enzymes with exquisite specificity and that utilize an often-exotic array of chemical substrates. In no molecule are modifications more prevalent than in transfer RNAs. In the present document, we will attempt to take a chemical rollercoaster ride from prebiotic times to the present, with nucleoside modifications as key players and tRNA as the centerpiece that drove the evolution of biological systems to where we are today. These ideas will be put forth while touching on several examples of tRNA modification enzymes and their modus operandi in cells. In passing, we submit that the choice of tRNA is not a whimsical one but rather highlights its critical function as an essential invention for the evolution of protein enzymes. Keywords: tRNA modification; evolution; genetic code 1. Before There Was RNA, There Were Modifications Through the years there has been a seemingly limitless and constant supply of ideas and discussions about the origin of life; all envision numerous scenarios with the common theme that, based on modern day biological systems, replication and information storage were needed for a self-sustaining system to survive. -
Product Sheet Info
Master Clone List for NR-19280 Yersinia pestis Strain KIM Gateway® Clone Set, Recombinant in Escherichia coli, Plates 1-43 Catalog No. NR-19280 Table 1: Yersinia pestis Gateway® Clone, Plate 1 (UYPVA), NR-195971 Clone Well Locus ID Description ORF Accession Average Position Length Number Depth of Coverage 38250 A01 NTL02YP3655 transcriptional activator of nhaA 900 AAM87251.1 3.90531915 38268 A02 NTL02YP0999 apbA protein 912 AAM84595.1 6.01785714 38344 A03 NTL02YP3653 putative regulator 939 AAM87249.1 5.6680286 38375 A04 NTL02YP3668 homoserine kinase 951 AAM87264.1 5.57214934 38386 A05 NTL02YP1006 cytochrome o ubiquinol oxidase subunit II 957 AAM84602.1 5.45937813 38408 A06 NTL02YP2211 suppressor of htrB, heat shock protein 963 AAM85807.1 5.40877368 38447 A07 NTL02YP3649 penicillin tolerance protein 978 AAM87245.1 5.34086444 38477 A08 NTL02YP0988 thiamin-monophosphate kinase 990 AAM84584.1 6.8223301 36045 A09 NTL02YP3670 hypothetical protein 159 AAM87266.1 4.53266332 36094 A10 NTL02YP3672 hypothetical protein 174 AAM87268.1 4.72429907 36136 A11 NTL02YP1000 hypothetical protein 189 AAM84596.1 5.25327511 36241 A12 NTL02YP2577 hypothetical protein 225 AAM86173.1 3.88301887 membrane-bound ATP synthase, F0 36280 B01 NTL02YP4086 240 AAM87682.1 4.79285714 sector, subunit c 36342 B02 NTL02YP3654 30S ribosomal subunit protein S20 264 AAM87250.1 3.86513158 36362 B03 NTL02YP2570 hypothetical protein 270 AAM86166.1 5.40322581 38504 B04 NTL02YP2573 putative ABC transporter permease 996 AAM86169.1 6.65444015 38516 B05 NTL02YP0234 putative heat shock