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MOLECULAR MECHANISM of the TRAMP COMPLEX by HUIJUE JIA

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MOLECULAR MECHANISM of the TRAMP COMPLEX by HUIJUE JIA MOLECULAR MECHANISM OF THE TRAMP COMPLEX by HUIJUE JIA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation advisor: Dr. Eckhard Jankowsky Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY August, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________Huijue Jia_____________________ candidate for the ______Doctor of Philosophy______ degree* . (signed)_____________William C. Merrick________________ (chair of the committee) _____________Eckhard Jankowsky________________ _____________Michael E. Harris__________________ _____________Jeff Coller_______________________ _____________Alan M. Tartakoff_________________ (date) _________________June 3, 2011__________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Dedication ―But lately it seems to me that dedicating a book is like the fine rhetoric about offering one's life to one's country, or handing the reins of the government back to the people. This is but the vain and empty juggling of language. Despite all the talk about handing it over, the book remains like the flying knife of the magician—released without ever leaving the hand. And when he dedicates his work in whatever manner he chooses, the work is still the author's own. Since my book is a mere trifle, it does not call for such ingenious disingenuousness. I therefore have not bothered myself about the dedication.‖ Quoted from the preface of Fortress Beseiged by Qian Zhongshu (Ch’ien Chung- shu). Translated by Jeanne Kelly and Nathan K. Mao. Table of contents List of tables.......................................................................................................................... viii List of figures .......................................................................................................................... ix Acknowledgements ............................................................................................................... xv List of abbreviations ............................................................................................................ xvi Abstract ................................................................................................................................. xix Chapter 1: General introduction to SF2 RNA helicases ..................................................... 1 1.1 Introduction to RNA helicases ..................................................................................... 1 1.2 Classification of helicases ............................................................................................. 2 1.3 Sequence and structural features of SF2 RNA helicases ........................................... 3 1.3.1 Conserved sequence motifs in the helicase core ...................................................... 3 1.3.2 N- and C-terminal domains ...................................................................................... 7 1.3.3 β-hairpin structure .................................................................................................... 8 1.4 Unwinding mechanism of SF2 RNA helicases ............................................................ 9 1.5 Activities of SF2 RNA helicases with relationship to cellular functions ................ 11 1.5.1 Diverse cellular functions of SF2 RNA helicases.................................................. 11 1.5.2 RNA duplex unwinding ......................................................................................... 12 1.5.3 Protein displacement .............................................................................................. 14 1.5.4 RNA chaperone activity ......................................................................................... 15 1.5.5 RNA clamps and protein binding platform ............................................................ 16 1.6 SF2 RNA helicases and their cofactors ..................................................................... 16 i 1.7 Studies on TRAMP and Ded1p in this thesis ........................................................... 18 1.7.1 The Ski2-like protein Mtr4p in the TRAMP complex ........................................... 18 1.7.2 The DEAD-box proteins Ded1p ............................................................................ 19 Chapter 2: Introduction--The TRAMP complex, a key player in nuclear RNA metabolism ........................................................................................................ 21 2.1 Components of the TRAMP complex and their biochemical activities ................. 21 2.1.1 The TRAMP complex ............................................................................................ 21 2.1.2 Difference between TRAMP- and canonical PAP-mediated polyadenylation ...... 22 2.1.3 The poly(A) polymerases Trf4p and Trf5p ............................................................ 23 2.1.4 The zinc-knuckle proteins Air1p and Air2p .......................................................... 27 2.1.5 The Ski2-like helicase Mtr4p ................................................................................. 28 2.2 The 3’->5’ exonuclease complex exosome ................................................................. 32 2.3 Difference between the TRAMP/exosome pathway and the bacterial degradosome ....................................................................................................................................... 33 2.4 RNA substrates of the TRAMP complex .................................................................. 35 2.4.1 Ribosomal RNAs (rRNAs) .................................................................................... 35 2.4.2 Small nucleolar RNAs (snoRNAs) ........................................................................ 36 2.4.3 Transfer RNAs (tRNAs) ........................................................................................ 36 2.4.4 Small nuclear RNAs (snRNAs) ............................................................................. 37 2.4.5 Long transcripts of RNA polymerase II (Pol II) .................................................... 37 2.4.6 Mechanism of substrate identification by TRAMP is unknown ............................ 38 2.5 TRAMP and chromatin maintenance ....................................................................... 38 2.7 Biochemical characterization of the TRAMP complex ........................................... 45 ii Chapter 3: The RNA helicase Mtr4p modulates polyadenylation in the TRAMP complex .............................................................................................................. 45 3.1 Introduction ................................................................................................................. 46 3.2 Results .......................................................................................................................... 46 3.2.1 Reconstitution and purification of the S. cerevisiae TRAMP complex from proteins expressed in E. coli................................................................................... 46 3.2.2 TRAMP displays modulated polyadenylation activity .......................................... 47 Met 3.2.3 Hypomethylated pre-tRNAi , a prototypical TRAMP target, accumulates poly(A) tails with approximately 4 adenylates in vivo ........................................................ 53 3.2.4 Modulated polyadenylation activity with generic model substrates ...................... 56 3.2.5 The modulation of polyadenylation activity depends on Mtr4p ............................ 60 3.2.6 The modulation of polyadenylation activity depends on the number of 3’-terminal adenylates ............................................................................................................... 65 3.2.7 Residues outside the helicase domain of Mtr4p participate in the detection of 3’- terminal adenylates................................................................................................. 66 3.2.8 Generation of short poly(A) tails involves multiple cycles of TRAMP binding and dissociation ............................................................................................................. 69 3.2.9 Mtr4p modulates Trf4p activity through multiple, energetically small effects ..... 72 3.3 Discussion..................................................................................................................... 74 3.3.1 A key role for Mtr4p in the regulation of poly(A) tail lengths for RNAs processed by TRAMP ............................................................................................................. 74 3.3.2 A new type of function for an RNA helicase: control of another enzyme in iii response to features in the RNA ............................................................................ 75 3.3.3 Direct interrogation of 3’-terminal bases by Mtr4p ............................................... 76 Chapter 4: Stimulation of the RNA helicase Mtr4p in the TRAMP complex ................. 79 4.1 Introduction ................................................................................................................. 79 4.2 Results .......................................................................................................................... 80 4.2.1 Trf4p/Air2p in the TRAMP complex stimulates the unwinding activity of Mtr4p .....................................................................................................................
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