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Amino Acid Specificity in Translation
Opinion TRENDS in Biochemical Sciences Vol.30 No.12 December 2005 Amino acid specificity in translation Taraka Dale and Olke C. Uhlenbeck Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA Recent structural and biochemical experiments indicate For example, in the course of deducing the recognition that bacterial elongation factor Tu and the ribosomal rules of aaRSs, several amber-suppressor tRNA bodies A-site show specificity for both the amino acid and the were deliberately mutated such that they were amino- tRNA portions of their aminoacyl-tRNA (aa-tRNA) acylated by a different aaRS, and the resulting ‘identity- substrates. These data are inconsistent with the swapped’ tRNAs were shown to insert the new amino acid traditional view that tRNAs are generic adaptors in into protein [7,8]. In addition, suppressor tRNAs esterified translation. We hypothesize that each tRNA sequence with O30 different unnatural amino acids have been has co-evolved with its cognate amino acid, such that all successfully incorporated into protein [9]. Together, these aa-tRNAs are translated uniformly. data suggest that the translational apparatus lacks specificity for different amino acids, once they are Introduction esterified onto tRNA. The mechanism of protein synthesis is traditionally In a few isolated cases, however, the translation considered to have two phases with different specificities machinery seems to show specificity for the esterified towards the 20 amino acid side chains (Figure 1). In the amino acid. A prominent example occurs in the transami- first phase, each amino acid is specifically recognized by dation pathway, which is used as an alternative to GlnRS its cognate aminoacyl-tRNA synthetase (aaRS) and to produce Gln-tRNAGln in many bacteria and archaea esterified to the appropriate tRNA to form an aminoacyl- [10,11]. -
EF-Tu and Rnase E Are Functionally Connected
Till min familj List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Hammarlöf, D.L., Hughes, D. (2008) Mutants of the RNA- processing enzyme RNase E reverse the extreme slow-growth phenotype caused by a mutant translation factor EF-Tu. Molecular Microbiology, 70(5), 1194-1209 II †Bergman, J., †Hammarlöf, D.L., Hughes D. (2011) Reducing ppGpp levels rescues the extreme growth defect of mutant EF-Tu. Manuscript III Hammarlöf, D.L., Liljas, L., Hughes, D. (2011) Temperature- sensitive mutants of RNase E in Salmonella enterica. Journal of Bacteriology. In press IV †Hammarlöf, D.L., †Bergman, J., Hughes, D. (2011) Extragenic suppressors of RNase E. Manuscript †These authors contributed equally. Reprints were made with permission from the respective publishers. Contents Introduction ................................................................................................... 11 Bacterial growth ....................................................................................... 11 Translation in bacteria .............................................................................. 12 The ribosome ....................................................................................... 12 The translation cycle ............................................................................ 13 Elongation Factor Tu ................................................................................ 14 The most abundant protein in the cell ................................................. -
Archaeal Translation Initiation Revisited: the Initiation Factor 2 and Eukaryotic Initiation Factor 2B ␣--␦ Subunit Families
Proc. Natl. Acad. Sci. USA Vol. 95, pp. 3726–3730, March 1998 Evolution Archaeal translation initiation revisited: The initiation factor 2 and eukaryotic initiation factor 2B a-b-d subunit families NIKOS C. KYRPIDES* AND CARL R. WOESE Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences, MC 110, 407 S. Goodwin, Urbana, IL 61801 Contributed by Carl R. Woese, December 31, 1997 ABSTRACT As the amount of available sequence data bacterial and eukaryotic translation initiation mechanisms, increases, it becomes apparent that our understanding of although mechanistically generally similar, were molecularly translation initiation is far from comprehensive and that unrelated and so had evolved independently. The Methano- prior conclusions concerning the origin of the process are coccus jannaschii genome (7–9), which gave us our first wrong. Contrary to earlier conclusions, key elements of trans- comprehensive look at the componentry of archaeal transla- lation initiation originated at the Universal Ancestor stage, for tion initiation, revealed that archaeal translation initiation homologous counterparts exist in all three primary taxa. showed considerable homology with eukaryotic initiation, Herein, we explore the evolutionary relationships among the which, if anything, reinforced the divide between bacterial components of bacterial initiation factor 2 (IF-2) and eukary- initiation and that seen in the other domains. We recently have otic IF-2 (eIF-2)/eIF-2B, i.e., the initiation factors involved in shown, however, that bacterial translation initiation factor 1 introducing the initiator tRNA into the translation mecha- (IF-1), contrary to previously accepted opinion, is related in nism and performing the first step in the peptide chain sequence to its eukaryotic/archaeal (functional) counterpart, elongation cycle. -
Initiation Factor Eif5b Catalyzes Second GTP-Dependent Step in Eukaryotic Translation Initiation
Initiation factor eIF5B catalyzes second GTP-dependent step in eukaryotic translation initiation Joon H. Lee*†, Tatyana V. Pestova†‡§, Byung-Sik Shin*, Chune Cao*, Sang K. Choi*, and Thomas E. Dever*¶ *Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-2716; ‡Department of Microbiology and Immunology, State University of New York Health Science Center, Brooklyn, NY 11203; and §A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Edited by Harry F. Noller, University of California, Santa Cruz, CA, and approved October 31, 2002 (received for review September 19, 2002) Initiation factors IF2 in bacteria and eIF2 in eukaryotes are GTPases In addition, when nonhydrolyzable GDPNP was substituted Met that bind Met-tRNAi to the small ribosomal subunit. eIF5B, the for GTP, eIF5B catalyzed subunit joining; however, the factor eukaryotic ortholog of IF2, is a GTPase that promotes ribosomal was unable to dissociate from the 80S ribosome after subunit subunit joining. Here we show that eIF5B GTPase activity is re- joining (7). quired for protein synthesis. Mutation of the conserved Asp-759 in To dissect the function of the eIF5B G domain and test the human eIF5B GTP-binding domain to Asn converts eIF5B to an model that two GTP molecules are required in translation XTPase and introduces an XTP requirement for subunit joining and initiation, we mutated conserved residues in the eIF5B G translation initiation. Thus, in contrast to bacteria where the single domain and tested the function of the mutant proteins in GTPase IF2 is sufficient to catalyze translation initiation, eukaryotic translation initiation. -
João Cancela De Amorim Falcão Paredes Estudo Molecular Da
Universidade de Aveiro Departamento de Biologia 2010 João Cancela de Estudo molecular da degeneração e evolução Amorim Falcão celular induzidas por erros na tradução do mRNA Paredes Molecular study of cell degeneration and evolution induced by mRNA mistranslation Universidade de Aveiro Departamento de Biologia 2010 João Cancela de Estudo molecular da degeneração e evolução Amorim Falcão celular induzidas por erros na tradução do mRNA Paredes Molecular study of cell degeneration and evolution induced by mRNA mistranslation Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Biologia, realizada sob a orientação científica do Doutor Manuel António da Silva Santos, Professor Associado do Departamento de Biologia da Universidade de Aveiro. Apoio financeiro do POCI 2010 no âmbito do III Quadro Comunitário de Apoio, comparticipado pelo FSE e por fundos nacionais do MCES/FCT. “The known is finite, the unknown infinite; intellectually we stand on an islet in the midst of an illimitable ocean of inexplicability. Our business in every generation is to reclaim a little more land, to add something to the extent and the solidity of our possessions” Thomas Henry Huxley (1825 – 1895) o júri presidente Doutor Domingos Moreira Cardoso Professor Catedrático da Universidade de Aveiro Doutora Claudina Amélia Marques Rodrigues Pousada Professora Catedrática Convidada da Universidade Nova de Lisboa Doutor António Carlos Matias Correia Professor Catedrático da Universidade de Aveiro Doutor -
Mitochondrial Translation and Beyond: Processes Implicated in Combined Oxidative Phosphorylation Deficiencies
Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2010, Article ID 737385, 24 pages doi:10.1155/2010/737385 Review Article Mitochondrial Translation and Beyond: Processes Implicated in Combined Oxidative Phosphorylation Deficiencies Paulien Smits, Jan Smeitink, and Lambert van den Heuvel Department of Pediatrics, Nijmegen Center for Mitochondrial Disorders, Radboud University Nijmegen Medical Center, Geert Grooteplein 10, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Correspondence should be addressed to Lambert van den Heuvel, [email protected] Received 31 October 2009; Accepted 29 January 2010 Academic Editor: Aikaterini Kontrogianni-Konstantopoulos Copyright © 2010 Paulien Smits et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders. -
Open FINAL GRAD SCHOOL.Pdf
The Pennsylvania State University The Graduate School Eberly College of Science PHOSPHOPROTEOMIC ANALYSIS OF RIBOSOMAL PROTEINS: IMPLICATIONS IN TRANSLATION AND APOPTOSIS A Dissertation in Biochemistry, Microbiology, and Molecular Biology by Jennifer Lynn Miller © 2009 Jennifer Lynn Miller Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2009 The dissertation of Jennifer Lynn Miller was reviewed and approved* by the following: Emine C. Koc Assistant Professor Biochemistry and Molecular Biology Dissertation Advisor Chair of Committee Robert A. Schlegel Professor of Biochemistry and Molecular Biology Wendy Hanna-Rose Assistant Professor Biochemistry and Molecular Biology Ming Tien Professor of Biochemistry Erin D. Sheets Assistant Professor of Chemistry Richard J. Frisque Professor of Molecular Virology Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School. ABSTRACT Mammalian mitochondrial ribosomes synthesize thirteen proteins that are essential for oxidative phosphorylation. Besides having a major role in ATP synthesis, mitochondria also contribute to biochemical processes coordinating apoptosis, mitochondrial diseases, and aging in eukaryotic cells. This unique class of ribosomes is protein-rich and distinct from cytoplasmic ribosomes. However, mitochondrial ribosomes (55S) share a significant homology to bacterial ribosomes (70S), particularly in size, the general mechanism of translation, and ribosomal protein content. Due to the overall resemblance between the two systems and the earlier reports of post-translational modifications, we investigated how phosphorylation of ribosomal proteins from bacteria and mitochondria regulates translation and other acquired roles. Identification of twenty- four phosphorylated 70S and 55S ribosomal proteins as well as the potential endogenous kinase was achieved using 2D-gel electrophoresis and tandem mass spectrometry. -
Trans-Acting Factors Affecting Retroviral Recoding
TRANS-ACTING FACTORS AFFECTING RETROVIRAL RECODING Lisa Green Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2012 © 2012 Lisa Green All Rights Reserved Thesis Abstract Trans-Acting Factors Affecting Retroviral Recoding Lisa Green The production of retroviral enzymes requires a translational recoding event which subverts normal decoding, either by direct suppression of termination with the insertion of an amino acid at a stop codon (readthrough), or by an alteration of the reading frame of the mRNA (frameshift). It has been determined that retroviral readthrough and frameshift require cis-acting factors in the mRNA to stimulate recoding on the eukaryotic ribosome. Here we investigate the affects of trans-acting factors on recoding, primarily in the context of the MoMLV gag-pol junction. We report the effects of a host protein, Large Ribosomal Protein Four (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we show that transfection of cells with an RPL4 cDNA expression construct enhances recoding efficiency in a dose-dependent manner. The increase in the frequency of recoding can be more than 2-fold, adequate to disrupt normal viral production. This effect is cell line specific, and appears to be distinct to RPL4 among ribosomal proteins. The RPL4 increase occurs with both retroviral readthrough and frameshift sequences, and even at other viral readthrough regions that do not involve RNA secondary structures. We show that RPL4 effects are negated by release factor over-expression, and that RPL4 will increase readthrough above the levels of a hyperactive mutant and in addition to G418. -
The Pennsylvania State University the Graduate School Eberly
The Pennsylvania State University The Graduate School Eberly College of Science REGULATION OF MITOCHONDRIAL TRANSLATION AND OXIDATIVE PHOSPHORYLATION THROUGH REVERSIBLE ACETYLATION A Dissertation in Biochemistry, Microbiology and Molecular Biology by Hüseyin Çimen 2012 Hüseyin Çimen Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2012 The Dissertation of Hüseyin Çimen was reviewed and approved* by the following: Emine C. Koc Assistant Professor of Biochemistry and Molecular Biology Dissertation Co-adviser Co-chair of Committee Hasan Koc Assistant Professor of Natural Sciences Dissertation Co-adviser Co-chair of Committee Craig E. Cameron Paul Berg Professor of Biochemistry and Molecular Biology Associate Department Head for Research and Graduate Education Joseph C. Reese Professor of Biochemistry and Molecular Biology Teh-hui Kao Professor of Biochemistry and Molecular Biology Tae-Hee Lee Assistant Professor of Chemistry and the Huck Institute of the Life Sciences Craig E. Cameron Paul Berg Professor of Biochemistry and Molecular Biology Associate Department Head of the Department of Biochemistry and Molecular Biology iii ABSTRACT In a eukaryotic cell, mitochondria provide energy in the form of ATP through oxidative phosphorylation (OXPHOS), which consists of five electron transport chain complexes embedded in the inner membrane of mitochondria. Human mitochondria have their own genome and transcription/translation system to synthesize mitochondrially encoded thirteen proteins of respiratory chain complexes. We investigated how acetylation of ribosomal proteins regulates translation and energy production in mitochondria since reversible acetylation of mitochondrial proteins was found to be critical for maintaining energy homeostasis. We identified mitochondrial ribosomal protein L10 (MRPL10) as the major acetylated ribosomal protein in mammalian mitochondria with two-dimensional gel electrophoresis followed by tandem mass spectrometry and immunoblotting analyses. -
The Carboxyl Termini of RAN Translated GGGGCC Nucleotide Repeat Expansions Modulate Toxicity in Models of ALS/FTD Fang He1,2*, Brittany N
He et al. Acta Neuropathologica Communications (2020) 8:122 https://doi.org/10.1186/s40478-020-01002-8 RESEARCH Open Access The carboxyl termini of RAN translated GGGGCC nucleotide repeat expansions modulate toxicity in models of ALS/FTD Fang He1,2*, Brittany N. Flores1, Amy Krans1, Michelle Frazer1,3, Sam Natla1, Sarjina Niraula2, Olamide Adefioye2, Sami J. Barmada1 and Peter K. Todd1,4* Abstract An intronic hexanucleotide repeat expansion in C9ORF72 causes familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This repeat is thought to elicit toxicity through RNA mediated protein sequestration and repeat-associated non-AUG (RAN) translation of dipeptide repeat proteins (DPRs). We generated a series of transgenic Drosophila models expressing GGGGCC (G4C2) repeats either inside of an artificial intron within a GFP reporter or within the 5′ untranslated region (UTR) of GFP placed in different downstream reading frames. Expression of 484 intronic repeats elicited minimal alterations in eye morphology, viability, longevity, or larval crawling but did trigger RNA foci formation, consistent with prior reports. In contrast, insertion of repeats into the 5′ UTR elicited differential toxicity that was dependent on the reading frame of GFP relative to the repeat. Greater toxicity correlated with a short and unstructured carboxyl terminus (C-terminus) in the glycine-arginine (GR) RAN protein reading frame. This change in C-terminal sequence triggered nuclear accumulation of all three RAN DPRs. A similar differential toxicity and dependence on the GR C-terminus was observed when repeats were expressed in rodent neurons. The presence of the native C-termini across all three reading frames was partly protective. -
RPS25 Is Required for Efficient RAN Translation of C9orf72 and Other Neurodegenerative Disease-Associated Nucleotide Repeats
BRIEF COMMUNICATION https://doi.org/10.1038/s41593-019-0455-7 RPS25 is required for efficient RAN translation of C9orf72 and other neurodegenerative disease-associated nucleotide repeats Shizuka B. Yamada1,2, Tania F. Gendron 3, Teresa Niccoli4,5,6, Naomi R. Genuth1,2,7, Rosslyn Grosely8, Yingxiao Shi9, Idoia Glaria4,5, Nicholas J. Kramer 1,10, Lisa Nakayama1, Shirleen Fang1, Tai J. I. Dinger1,2, Annora Thoeng4,5,6, Gabriel Rocha9, Maria Barna1,7, Joseph D. Puglisi8, Linda Partridge 6, Justin K. Ichida9, Adrian M. Isaacs 4,5, Leonard Petrucelli3 and Aaron D. Gitler 1* Nucleotide repeat expansions in the C9orf72 gene are the ATG-initiated green fluorescent protein (GFP) construct. We identi- most common cause of amyotrophic lateral sclerosis and fied 42 genes that either increased or decreased DPR levels without sim- frontotemporal dementia. Unconventional translation (RAN ilarly regulating ATG–GFP (Fig. 1c and see Supplementary Fig. 1a–c). translation) of C9orf72 repeats generates dipeptide repeat We also performed quantitative PCR with reverse transcription proteins that can cause neurodegeneration. We performed (RT–qPCR) to identify hits that affected transcription or RNA stabil- a genetic screen for regulators of RAN translation and iden- ity of the repeat RNA (see Supplementary Table 1). tified small ribosomal protein subunit 25 (RPS25), pre- One striking hit from the screen was the deletion of RPS25A, senting a potential therapeutic target for C9orf72-related which encodes a eukaryotic-specific, non-essential protein compo- amyotrophic lateral sclerosis and frontotemporal dementia nent of the small (40S) ribosomal subunit11,12. RPS25 plays a criti- and other neurodegenerative diseases caused by nucleotide cal role in several forms of unconventional translation including repeat expansions. -
Poster Session 10: Translation 21:00 - 22:00 Friday, 29Th May, 2020 Poster
Poster Session 10: Translation 21:00 - 22:00 Friday, 29th May, 2020 Poster 66 Translational fidelity is maintained through precise aminoacyl-tRNA accommodation dynamics gated by Elongation Factor Tu Dylan Girodat1, Scott Blanchard2, Hans-Joachim Wieden3, Karissa Sanbonmatsu1 1Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, USA. 2Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. 3Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Alberta, Canada Abstract The fidelity of translation is enigmatic, as the efficiency of cognate aminoacyl(aa)-tRNA selection by the ribosome is greater than what can be predicted from Watson-Crick base-pairing between the codon in the mRNA and the anticodon in the tRNA. The complexity of this process arises from the fact that aa-tRNA selection is a multistep process aided by auxiliary proteins such as the GTPase elongation factor (EF)-Tu, responsible for delivery of aa-tRNA to the ribosome. As such, the precise structural mechanism of how the ribosome in complex with EF-Tu selects for cognate aa-tRNA remains to be fully resolved. Here, using all-atom molecular dynamics (MD) simulations, we identify subtle differences between cognate and near-cognate aa-tRNA movement into the ribosome and how conformational rearrangements of EF-Tu aid in tRNA selection. Near-cognate aa-tRNA accommodation follows an alternative trajectory, compared to cognate aa-tRNA, leading to a misaligned position within the A-site. The origins of the alternative trajectory originate from the perturbed base-pairing between the codon and anticodon of the mRNA and tRNA, respectively.