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Maturation of Trna in Haloferax Volcanii. Dissertation Presented In Maturation of tRNA in Haloferax volcanii. Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Richard Neil Nist BA, MS Graduate Program in Microbiology The Ohio State University 2011 Dissertation Committee: Professor Charles J. Daniels, Advisor Associate Professor Juan Alfonzo Professor Venkat Gopalan Copyright by Richard Neil Nist 2011 Abstract Transfer RNA maturation in Archaea is a multi-faceted process requiring the activities of numerous enzymes in order to generate functional tRNAs from primary transcripts. Upon transcription, ribonuclease P cleaves the 5’ leader, tRNase Z cleaves the 3’ trailer, RNA-dependent and RNA-independent enzymes introduce modifications to bases and sugars, 3’ maturation is completed by the CCA-adding enzyme, and introns are removed. We examined the modification and intron removal stages of maturation for the Haloferax volcanii intron-containing pre-tRNA. H. volcanii tRNAMet possesses a 2’-O-methyl modification at position C34. A similar modification was characterized in pre-tRNATrp as being guided by a box C/D sRNP complex. Furthermore, just as sequence analysis and in vitro experiments have shown the intron-containing pre-tRNATrp to be a substrate for box C/D-guided modification, sequence analysis of intron-containing pre-tRNAMet suggests it is a substrate for box C/D-guided modification. We have determined not only that the C34 modification of tRNAMet is guided by a box C/D sRNP, but also that the intron- containing pre-tRNAMet appears to be a modification substrate in vivo. By the action of intron endonuclease, tRNA introns are removed in Archaea. This is a requisite step for the maturation of tRNATrp, tRNAMet, and tRNAGlnUUG in H. volcanii. Sequence analysis and previous mapping experiments suggest that there are additional substrates for this enzyme, including the 16S rRNA and 23S rRNA flanking ii sequences. Our data indicate that the 16S rRNA and 23S rRNA flanking sequences are cleaved by the intron endonuclease meaning there are substrates beyond the three tRNAs. Ribosomal protein L7ae has been at the forefront or background of each study conducted in this research. This K-turn stabilizing protein is involved in several facets of tRNA maturation including as a box C/D sRNP complex component required for 2’-O- methylation of tRNATrp and tRNAMet, as a component of archaeal RNase P, and as a box C/D sRNP complex component for the putative guide sRNA formed by the ligation of the 23S rRNA flanking sequences. Given the involvement of L7ae in these and other sRNP complexes, we attempted to capture L7ae-containing complexes from H. volcanii. We were able to demonstrate L7ae co-purification with each known and predicted class of L7ae-binding RNAs. iii Dedication To those who provided support/assistance for finishing the job, you know who you are. iv Acknowledgements This amount of work could never have been accomplished without considerable assistance from others. First, I would like to thank my advisor Dr. Chuck Daniels for allowing me to pick up and move this interesting project along. The back-and-forth discussions of data and suggestions of how to proceed next were greatly beneficial providing both insights I had not thought of and aiding me in seeing all sides of the problem. Also, the atmosphere of encouragement and continued interest in the science helped reinforce my own views. Gratitude also goes out to my advisory and thesis committee, Dr. Juan Alfonzo, Dr. Kurt Fredrick, Dr. Joe Krzycki, and Dr. Venkat Gopalan for their willingness to share their insights into the research problems and lab protocols for similar procedures. The additional perspectives provided by them were significantly helpful in seeing likely explanations and problems that I otherwise would have missed. The science may be the focus, but the people really make the lab. That said, thanks to my long-time lab-mates Dr. Anice Sabag-Daigle and Shoko Morimoto; Anice for being a willing sounding board for great discussions about our work and firming up protocols and Shoko for sharing protocols and keeping those buffer mixes handy that I always seemed to need. I would also be remiss if I didn’t thank them both (as well as past lab members Brian Gabel and Kara Batte) for putting up with me throughout these years, and making this a good working environment. I also thank past members of the lab whose contributions led to the project I was given. v I am appreciative of the assistance of our collaborators on the tRNAMet sRNA project provided by Dr. Jens Hemmingsen and his group at Capital University (Columbus, Ohio). In particular for the development of the RNase H assay which finally allowed us to “close the book” on the tRNAMet sRNA project. I am also appreciative of the understanding from the TA supervisors, allowing me to balance out teaching and research, and the friendly assistance of the other graduate students in the program at OSU, there are too many of you to name, but you have my thanks for our work and non- work related discussions. Lastly, but hardly least, I wish to express my gratitude to my family, in particular to my parents Tim and Judy for their strong, constant support and interest throughout these last several years. Also, thanks to my brothers Jeff and Tom and sister Denise for their interest and attentive ears along the way, and to my grandparents Neil and Eris for their faith in me and support. vi Vita April 19, 1982 ...................................................................................... Born – Canton, Ohio 2004............................................................................................................. Bachelors of Art Capital University Columbus, Ohio 2007......................................................................................................... Masters of Science The Ohio State University Columbus, Ohio 2007-present ..................................................... Graduate Teaching and Research Associate The Ohio State University Columbus, Ohio Field of Study Major Field: Microbiology vii Table of Contents Page Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgements ..............................................................................................................v Vita ................................................................................................................................... vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii List of Abbreviations ....................................................................................................... xvi Chapter 1: Archaea and Stable RNAs Domain Archaea .........................................................................................................1 Stable RNAs ...............................................................................................................5 Research Focus ........................................................................................................14 Chapter 2: Materials and Methods Materials and Enzymes .............................................................................................16 Culturing of Bacteria and Archaea ...........................................................................17 Culturing of Escherichia coli .......................................................................17 Culturing of Haloferax volcanii ...................................................................21 Preparation of freezer stocks ........................................................................21 Culturing of H. volcanii strains in competition ............................................21 Nucleic acid preparations ........................................................................................22 RNA isolation ..............................................................................................22 H. volcanii genomic DNA isolation .............................................................24 Plasmid mini-preps ......................................................................................25 Quantitation of DNA/RNA ..........................................................................26 viii Electrophoretic techniques for nucleic acids separation and purification ................26 Denaturing polyacrylamide gel electrophoresis of DNA and RNA.............26 Agarose gel electrophoresis of RNA............................................................27 Non-denaturing agarose gel electrophoresis of DNA ..................................28 Gel staining with ethidium bromide .............................................................28 Recovery of nucleic acids from agarose ......................................................29 Recovery of nucleic acids from polyacrylamide ..........................................29 UV-shadowing .............................................................................................30 Preparation of competent cells and transformation ..................................................30
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