Species specific differences in recognition of tRNA(Pro) by prolyl tRNA synthetases

INFORMATION TO USERS This manuscript has been reproduced from the microfilm master UMI films the text directly from th e original o r copy submitted Thus, som e thesis and dissertation copies are in typew riter face, while others m ay be from any type o f com puter printer The quality of this reproduction is dependent upon the quality of the copy submitted B roken o r indistinct print, colored o r poor quality illustrations and photographs, p rin t bleedthrough, substandard m argins, and improper alignm ent can adversely affect reproduction In the unlikely event th a t the author did not send UMI a com plete manuscript and there are m issing pages, these will be noted Also, if unauthorized copyright material had to be rem oved, a note will indicate the deletion Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning a t the upper left-hand co rn e r and continuing from left to right in equal sections w ith small overlaps Photographs included xerographically in this in the original manuscript have copy H igher quality 6” x 9" been reproduced black and white photographic prints are available fo r any photographs or illustrations appearing in this copy for an additional charge Contact UMI directly to order P ro Q uest Inform ation and Learning 300 North Z eeb Road, Ann Arbor, Ml 48106-1346 U S A 800-521-0600 Reproduced with permission of the copyright owner Further reproduction prohibited w ithout permission Reproduced with permission of the copyright owner Further reproduction prohibited without permission Species-Specific Differences in Recogniton of tRNA(Pro) by Prolyl-tRNA Synthetases A THESIS SUBMITTED TO TH E FACULTY OF TH E GRADUATE S C H O O L O F TH E UNIVERSITY OF MINNESOTA BY Brian William Burke IN PARTIAL FULFILLMENT OF TH E REQUIREMENTS FOR THE DEGREE O F DOCTOR OF PHILOSOPHY Karin Musier-Forsyth, A dvisor May, 2001 Reproduced with permission of the copyright owner Further reproduction prohibited without permission UMI Number: 3008671 Copyright 2001 by Burke, Brian William All rights reserved _ ® UMI UMI Microform 3008671 Copyright 2001 by Bell & Howell Information and Learning Company All rights reserved This microform edition is protected against unauthorized copying under Title 17, United States Code Bell & Howell Information and Learning Company 300 North Zeeb Road P.O Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner Further reproduction prohibited without permission © Brian W Burke 2001 Reproduced with permission of the copyright owner Further reproduction prohibited without permission UNIVERSITY O F MINNESOTA This is to certify that I have exam ined this copy of a doctoral thesis by Brian W illiam Burke and have found that it is complete and satisfactory in all respects, and th a t any and all revisions required by the final exam ining com m ittee have been made Karin Musier-Forsyth Name of Faculty Adviser Signature o f Faculty Adviser May 22, 2001 Date GRADUATE SCHOOL Reproduced with permission of the copyright owner Further reproduction prohibited without permission Preface The following works in this thesis have been previously published Chapter 2: Catherine Stehlin, Brian Burke, Fan Yang, Hongjian Liu, Kiyotaka Shiba and Karin Musier-Forsyth (1998) Species-Specific Differences in the Operational RNA Code for Aminoacylation o f tR N A Pro Biochem istry 37, 8605-8613 ©1998 American Chem ical Society Chapter 3: Brian Burke, Fan Yang, Fei Chen, Catherine Stehlin, Barden Chan and Karin MusierForsyth (2000) Evolutionary Co-Adaptation of the M otif 2-Acceptor Stem Interaction in the Class II Prolyl-tRNA Synthetase System B iochem istry 39, 15540-15547 ©2000 American Chem ical Society Chapter 4: Brian Burke, Richard S Lipman, Kiyotaka Shiba, Karin Musier-Forsyth and Ya-Ming Hou (2001) Divergent Adaptation of tRNA Recognition by Methanococcus jannaschii ProlyltRNA Synthetase J Biol Chem 27, in press ©2001 The American Society fo r Biochemistry and M olecular Biology i Reproduced with permission of the copyright owner Further reproduction prohibited without permission ACS PUBLICATIONS DIVISION GUIDELINES FOR THESES AND DISSERTATIONS ATTN: STUDENTS STUDENT ADVISORS, AND TEACHERS Permission is now autom atically granted to ndude^w rpapoO ;) or portions o f your paperfs) in your thess; 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Call ACS Publications Division Copyright Office staff at 202/872-4368, or e-mail us at copvright@acs.org A u g tu l 199S Reproduced with permission of the copyright owner Further reproduction prohibited without permission B n s Brake Dep« ctim t o f Cbeaistty ThnvzsityafMm iicaon 2071 iexscstSt.SE 139‘ td feH *fl MianbpoEt.M ysS4S5 Bhani: 612-624-0510 F A X 612-626-75+1 + M y y2Q01 Pbb&c B ia s Office Oopyqght «sd Besc A n n cm Society fbrTUochm iattj «dMDtocn]«rBintofy 96SDJ iccivifleKbe Bada *.M D 20814-3996 To'W I om itM ay O iw rn r I a m * m in j id j e q a n t p n a u a a n t o m e th e fa Z k n asg m e m e r i p t ( a f w i a d i I m s a t an th e r i n ia y d s n t t H s o ,w iA tb e B c d e x tn ^ c q g A n th e ie g iix n e d c o p y r ig h t c m d B t Sue w iH a j x a rtw flx th e t a m gMi f l t t t t t o p c r m w k i n a o n ly Her flic m g a c a a f la w d e Buxke; Bnaz,IkpmK&,Sk&«nlS^ Shiba Kyoaka.Masscx-Eoayih, Karin, and Hmoc.Y*^£ng (2001) D Ivexgeig Adaptation o f tRXA Recognaioa by ife jkanooocem i ftw n a td d i Prclyl-tRXA Syrffhrtmr Io to n o i o fB io lo g ic a l C h em istry, in pan a le a n t* reached by phone a t612-624-0510 and by FAX a t612-626-7541 or by e-na £Lat bcrfag chcm.nmn.ttfo Thank yon v n y much fa t yoor coaaideaiioo- Sincetey PERMISSION GRANTEID contingent upon obtaining that of the autfnof B rian Btake _ — (j X f f ) * * fcr the copyright owner M AY $ 0 T H E AMERiCAK SOCETY FOR BiOCHESnlSTHY MOLECULAR BKXOGY iii Reproduced with permission of the copyright owner Further reproduction prohibited without permission j Acknowledgments First o f all, I would like to thank m y advisor, Professor Karin Musier-Forsyth, for her years o f support, assistance, and advice She is an excellent exam ple of what an advisor should be She has pushed me to grow as a scientist and researcher with encouragement all along I am grateful that she gave me the opportunity to travel and experience another culture with my several trips to Grenoble, som ething most graduate students not get the chance to I would also like to thank my other committee members, Prof Marian Stankovich, Prof Greg Connell, Prof Edgar Arriaga, Prof George Barany and Prof Hal Swafford, for their participation in my graduate career I am also grateful to many members of the KMF lab fo r making w ork much more fun than would be expected I thank them fo r their helpful discussions and materials they have given me throughout the years Dr Catherine Stehlin taught me most of the procedures I have carried out in lab and about the “blah-blah” I am glad she was so patient with a new student with no biological lab experience Dr Barden Chan, Dr Tim Stello, and Dr Penny J Beuning will always bring back fond memories I can’t imagine a better set of graduate students to learn from They have done so m uch for me and I hope I carried m yself as a mentor and example to the younger students as well as they did for me Barden, otherwise know as the Eatus alotus, was always a wealth of information fo r me as well as a good source o f amusement I thank Stello for not only putting up with me in lab, but also as a roommate as well Penny (that is HRH PJB) has been a great labm ate as well as a good friend I am thankful for all the oligos she has had to make me since I don’t know how to use the synthesizer! I also w a n t to thank Minh Hong fo r being such a good Bob Special thanks also to Abbey Rosen for iv Reproduced with permission of the copyright owner Further reproduction prohibited without permission Semi-synthetic E coli tRNAs were prepared as previously described (37) To facilitate in vitro transcription, all 5'-3/4-length constructs lacked C1 as described previously (36) Phosphorothioate substitutions were incorporated into a synthetic 5'-15mer or 3'-16-mer oligonucleotide during automated chemical synthesis using the phosphoramidite method on a Gene Assem bler Special (Pharmacia) using EDITH in place of the oxidation step as described previously (35) These fragments were then heat annealed to a 3/4-length in vitro transcribed RNA fragm ent to form a semi-synthetic full-length E coli tRNAPro All RNAs were purified by 12% o r 16% denaturing polyacrylamide gel electrophoresis Phosphorothioate-containing oligos at positions 72 and 75 were separated using reversed-phase HPLC with a Beckman Ultrasphere C18 analytical column (0.46 x 25 cm) on a Beckman system monitored at 260 nm under conditions initially based on those in (39) Both oligonucleotides were separated at 1.0 ml/min with 0.1 M aqueous triethylammonium acetate (TEAA) pH 7.0 buffer and an acetonitrile gradient from 11-17% over a period of 20 The phophorothioate substitutions at position 73 and 75 were separated using the methods BURKE4X7 and BURKE407, respectively Diastereomers were identified by digestion with snake venom phosphodiesterase and nuclease P1 as described in (40) G.3.2.2 Enzyme Preparation Purification of histidine-tagged wild-type E coli ProRS was accomplished as described previously with plasmid pCS-M1S (41) ProRS concentrations were based on active-site titrations determined using the adenylate burst assay (14) 6.3.2.3 Aminoacylation with Proline Aminoacylation assays were carried out as previously described (41) 118 Reproduced with permission of the copyright owner Further reproduction prohibited without permission 6.3.3 Results and Discussion Single and multiple phosphorothioate substitutions were incorporated into the 5'