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Identification of Trna Modifications in T. Thermophilus: Wild Type HB8 and Mutant DTTHA1897 by LC-UV-MS/MS

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Identification of Trna Modifications in T. Thermophilus: Wild Type HB8 and Mutant DTTHA1897 by LC-UV-MS/MS Identification of tRNA modifications in T. thermophilus: wild type HB8 and mutant DTTHA1897 by LC-UV-MS/MS A Thesis submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science In the Department of Chemistry Of the College of Arts and Sciences by Lihua Fu Bachelor of Science from University of Science and Technology Beijing, China 2008 Abstract Transfer RNAs (tRNAs) are essential adapter molecules for translation of the genetic code into a peptide sequence at the ribosome. The study of tRNA modifications are essential subjects of bioscience and are waiting to be explored in the areas of identification, function, structure, biosynthesis and the sequence of tRNA, and the locations of the post-transcriptional modification on tRNA sequences. Thermus thermophilus wild type and ΔTTHA 1897 were the model system that used to peruse a better understanding on tRNA modifications in this work. TTHA1897 is a putative gidA gene, which is one of the two crucial biosynthesis enzyme genes for xm5s2U in E. coli. ΔTTHA 1897 is mutant strain with disruption of tRNA gene TTHA1897 in T. thermophilus. Growth phenotype studies including growth rate and temperature sensitivity studies demonstrated that ΔTTHA1897 experienced a slower growth rate and non-survival in minimal medium. An HPLC-UV-MS study identified mnm5s2U in the wild type but not in ΔTTHA1897. Taking advantages of FT-ICRMS, the unique fragmentation pattern of xm5s2U was discovered with a proposed mechanism. An effective SRM method was developed and tested using E. coli tRNA nucleosides based on the unique fragmentation pattern of xm5s2U. A triple quadrupole mass spectrometer was also used to investigate a neutral loss method for the identification of xm5s2U nucleosides. 1 List of Abbreviations RNA Ribonucleic Acid DNA Deoxyribonucleic Acid mRNA Messenger RNA tRNA Transfer RNA rRNA Ribosomal RNA T. thermophilus Thermus thermophilus E. coli Escherichia coli TEM Thermus Enhanced Media LC-MS Liquid Chromatography coupled with Mass Spectrometry ESI Electrospray ionization CID Collision induced dissociation MS/MS Tandem mass spectrometry SRM Select reaction monitoring MRM Multiple reaction monitoring FT-ICR Fourier transform ion cyclotron resonance A Adenine C Cytosine G Guanine U Uracil I Inosine cmnm5U 5-Carboxymethylaminomethyluridine 2 mnm5U 5-Methylaminomethyluridine nm5U 5-Aminomethyluridine cmnm5s2U 5- Carboxymethylaminomethyl-2-thiouridine mnm5s2U 5-Methylaminomethyl-2-thiouridine nm5s2U 5-Aminomethyl-2-thiouridine D dihydrouridine Ψ pseudouridine Q queuosine cmo5U uridine 5-oxyacetic acid acp3U 3-(3-amino-3-carboxypropyl)uridine mcm5U 5-methoxycarbonylmethyl-uridine m1G 1-methylguanosine m2G 2-methylguanosine m7G 7-methylguanosine Gm 2'-O-methylguanosine ac4C N4-acetylcytidine k2C lysidine Cm 2'-O-methylcytidine m5U 5-methyluridine Um 2'-O-methyluridine S2U 2-thiocytidine s4U 4-thiocytidine 3 m5s2U 5-methyl-2-thiouridine t6A N6-threonylcarbamoyladenosine ms2t6A 2-methylthio-N6-threonyl carbamoyladenosine m2A 2-methyladenosine m1A 1-methyladenosine m6A N6-methyladenosine io6A N6-(cis-hydroxyisopentenyl)adenosine 6 6 6 m 2A N , N –dimethyladenosine ms2i6A 2-methylthio-N6-isopentenyladenosine ms2io6A 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine i6A N6-isopentenyladenosine 4 Acknowledgement First of all, I would like to thank my advisor Dr. Limbach! He is one of the most supportive and smart teacher ever. I would never forget the moments of his kind, patience, and encouraging words when I was frustrated with my progress. Any time, he is always there to support and encourage. Especially, he guided me through the down times. I express my appreciation to him for his mentoring strategy. He always leaves so much room for me to discover through experimenting, but he is always there when I need help. I would like to send my heartfelt thanks to my committee members Dr. Heineman and De. Merino. Dr. Heineman’s kind words and guidance helps me very much on some daunting research subjects. Dr. Merino always helps me think about more aspects of my research. Without their long-term commitment of helping, I would never be able to go this far. A special thanks to Dr. Larry Sallans for his kindness and expertise while helping me with the instruments. I would like to thank the Limbach group members, Dr. Balu Addepalli, Dr. Kirk Gaston, Dr. Stephen Macha, Dr. Siwei Li, Dr. Romel Dator, Dr. Rebecca Rohlfs, Dr. Susan Russell, collin Wetzel, Wunan Shi, Xiaoyu Cao, Rob Ross, Yang Jiao. Dr. Gaston provided very valuable comments on many areas of my researches. I would also like to give special thanks to my friends, Dr. Tingting Wang, Dr. Li Duan, Wunan Shi, and Xiaoyu Cao for their continuous support during my time in University of Cincinnati. 5 I greatly appreciated continuous care and support from my family. My dearest parents and parents in law took excellent care of my children while I was working on test, research and teaching. My husband Dr. Xiaoping Chen is always there lending me a shoulder to rest. My two little girls are the most inspiring kids to mommy ever. 6 Table of Contents Abstract ........................................................................................................................................... 1 List of Abbreviations ........................................................................................................................ 2 Acknowdgement ............................................................................................................................. 5 Chapter 1. The Significance of tRNA ................................................................................................ 9 1.1 RNA Structure and Type. ....................................................................................................... 9 1.2 Transfer RNA Structure and Function .................................................................................. 10 1.3 tRNA Modifications and Their Significance ............................................................................. 10 1.4 Methods in Studying tRNA Modifications ........................................................................... 21 Chapter 2 Phenotype of T. Thermophilus HB8 Wild Type and ΔTTHA 1897 ................................. 23 2.1 Introduction ......................................................................................................................... 23 2.2 Materials and Methods ....................................................................................................... 23 2.2.1 T. thermophilus HB8 Wild Type .................................................................................... 24 2.2.2 The Construction of ΔTTHA1897 Disruption Mutant ................................................... 24 2.2.3 Total tRNA Isolation ...................................................................................................... 25 2.2.4 Growth Conditions ....................................................................................................... 26 2.3 Results and Discussion ........................................................................................................ 26 2.3.1 Total tRNA Isolation ...................................................................................................... 26 2.3.2 Phenotypes ................................................................................................................... 27 2.3.3 Discussion ..................................................................................................................... 29 2.4 Conclusions .......................................................................................................................... 30 Chapter 3 Identification of tRNA Modifications in T. thermophilus and Discovery of the Fragmentation Pattern of xm5s2U Nucleosides Family ................................................................ 31 3.1 Introduction ......................................................................................................................... 31 3.2 Materials and Methods ....................................................................................................... 31 3.2.1 Enzymatic Digestion ..................................................................................................... 31 _Toc425154930 3.2.2 HPLC-UV-LC/MS Analysis of Nucleosides ..................................................................... 32 3.2.3 Mass Condition ............................................................................................................. 34 3.3 Results and Discussion ........................................................................................................ 34 3.3.1 HPLC-UV-LCMS Analysis of Nucleosides ....................................................................... 34 3.3.2. Discussion .................................................................................................................... 37 7 3.4 Conclusions .......................................................................................................................... 38 Chapter 4. The Fragmentation Pattern of xm5s2U Nucleosides ................................................... 39 4.1 Introduction ......................................................................................................................... 39 4.2 Materials and Methods ......................................................................................................
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