STUDIES OF THE GENETIC ENCODING OF PYRROLYSINE FROM METHANOGENIC ARCHAEA DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Anirban Mahapatra, M.Sc. ***** The Ohio State University 2007 Dissertation Committee: Dr. Joseph A. Krzycki, Advisor Approved by Dr. Juan D. Alfonzo Dr. Charles J. Daniels Dr. Kurt L. Fredrick ___________________________ Advisor, Graduate Program in Microbiology ABSTRACT Pyrrolysine is the 22nd genetically encoded amino acid to be found in nature. Co-translational insertion at in-frame UAG codons proceeds so that a single pyrrolysine residue is found in the active site of all methylamine methyltransferases required for methylamine metabolism in Methanosarcina spp. This study examines processes central to the translation of UAG as pyrrolysine. The pylT gene encoding the tRNA for pyrrolysine, tRNAPyl, is part of the pyl operon of Methanosarcina spp. which also contains the pylS gene, encoding a class II aminoacyl-tRNA synthetase; and the pylB, pylC, and pylD genes proposed to catalyze the synthesis of pyrrolysine from metabolic precursors. Here, in the first study, by characterizing a Methanosarcina acetivorans mutant with the pylT gene region deleted, the essentiality of pyrrolysine incorporation in methanogenesis from methylamines is tested. The mutant lacks detectable tRNAPyl but grows similar to wild-type on methanol or acetate for which translation of UAG as pyrrolysine is likely not to be essential. However, unlike wild-type, the mutant can not grow on any methylamine or use monomethylamine as the sole source of nitrogen. Monomethylamine methyltransferase activity is detectable in wild-type cells, but not in mutant cells during growth on methanol. ii Further, the pyrrolysine-containing monomethylamine methyltransferase is absent from the mutant. This study is the first genetic analysis of UAG translation as pyrrolysine, and it reveals the phenotype of a Methanosarcina strain that can not decode UAG as pyrrolysine. PylS is an aminoacyl-tRNA synthetase encoded by the pylS gene adjacent to pylT. The data presented here in the second study shows that PylS catalyzes the ATP-dependent activation of synthetic pyrrolysine. PylS-catalyzed activation is highly specific for cognate amino acid, pyrrolysine, and does not require tRNA. Taken together with results obtained by others showing the ligation of pyrrolysine to tRNAPyl and the ability of pylS and pylT gene products to suppress amber codons in a recombinant system, the data presented indicates that PylS is a pyrrolysyl-tRNA synthetase capable of directly ligating pyrrolysine to tRNAPyl in vitro and in vivo. These results prove that PylS is the first aminoacyl-tRNA synthetase to be discovered from nature that is capable of attaching a genetically-encoded amino acid, not one of the common twenty, to cognate tRNA. Further, a metabolite from an Escherichia coli strain with the pylBCD genes expressed is shown to serve as a substrate for PylS activation. Along with results obtained by others showing PylS-catalyzed ligation of this metabolite to tRNAPyl, the data indicates that the pylB, pylC, and pylD gene products are sufficient for pyrrolysine synthesis from metabolic precursors common to M. acetivorans and E. coli. Earlier, in vitro studies by others indicated the two canonical lysyl-tRNA iii synthetases found in Methanosarcina spp. form a complex and slowly attach lysine to tRNAPyl. Owing to the implicit problem of substrate selection between lysine-tRNAPyl and pyrrolysine-tRNAPyl in translation, the relevance of the complex catalyzed ligation required testing in vivo. This is the focus of the third study. Data presented indicates that intact lysyl-tRNA synthetase genes are not required for methanogenesis from methylamines in Methanosarcina. Further, levels of monomethylamine methyltransferase and tRNAPyl aminoacylation are not diminished in lysyl-tRNA synthetase mutants. Taken together, the data presented here demonstrates that the indirect route of tRNAPyl aminoacylation involving complex formation of the two lysyl-tRNA synthetases is not essential for UAG translation as pyrrolysine. In the final study, the substrate specificity of PylS is probed using analogs of pyrrolysine. Features of the pyrroline ring of pyrrolysine that are determinants of PylS catalyzed activation are identified. Analogs used in functional probing in vitro are then incorporated in a monomethylamine methyltransferase in an Escherichia coli reporter system. iv Dedicated to the loving memory of my grandmother, Mahamaya Mahapatra v ACKNOWLEDGMENTS As I reflect on the last few years of my life, I feel a deep sense of gratitude to all who have made this work possible. First, I thank my advisor, Joseph Krzycki, for handing me a set of fascinating scientific problems to work on, and for patiently waiting as I developed the skills to analyze and interpret them. On a scientific level, he has been my guru in the original Sanskrit connotation of the word. I am also indebted to the members of my committee, Juan Alfonzo, Charles Daniels, Kurt Fredrick for their time and input on all aspects of my research and career development. Much of my work would have been delayed or simply, not possible without collaboration, and I thank Michael Chan and Bill Metcalf and members of their labs at Ohio State and the University of Illinois, Urbana-Champaign, respectively for supporting my project. I thank David Longstaff for befriending me when I knew no one else in the department. Jitesh Soares has become one of my closest friends, and I cannot thank him enough for always being there. I will always associate the best of times with Ross Larue’s boisterous laughter and I thank him heartily for his friendship through the years. I also thank Carey James, Sherry Blight, Jodie Lee, Ruisheng Jiang, Marsha Thalhofer, Jenee Smith, and Jess Dsyzel, among others, for making the ninth floor of the Riffe building such an agreeable work environment. vi Outside of work, I am grateful to Indrajit Mukherjee for sharing my sense of humor. I thank Rituparna Roy, Pushan Pahari, and Sudhakar Panda for the support over the years and miles. I thank my cousin, Prasun Pahari for being a co-conspirator in all creative endeavors, and my cousin, Atalanta Kar, for being a dependable “younger brother.” I am grateful to my in-laws - Tarasankar and Anjali Panigrahi; Deblina, and Nilanjana; and to my aunts - Jayasri Mahapatra, Asita Sarangi, and Manjusri Kar for their support. It is with sorrow that I remember personal losses. It is hard to believe my uncle, Prasanta Kar, is no more. I am inspired that I met such a staunchly idealistic person in a largely cynical world. I will deeply miss my uncle Arunangshu Panda and my aunt Sunita Acharya, and am grateful to have known them both. I am humbled when I realize that every single aspect of my life has been built on the sacrifices and prayers of my family members and well-wishers. I apologize for the impudence of thanking them, for without their support, I would be nothing. Theirs is a debt that I can never repay. My grandparents are not alive today, but I know their blessings are with me. My younger sister, Rita, has been a reliable source of common sense through the years. I am deeply grateful to my father for instilling an appreciation of the natural world. My mother has vii been a source of unconditional love my entire life, and I hope she is proud of how her son turned out. Last but not least, this work would never have been possible without the undying support of my wife, Rituparna, who sacrificed her own aspirations so that I could come to America to pursue mine. viii VITA December 3, 1974………………….Born, New Delhi, India 1997………………………………….B.Sc. (Hons) in Botany, Vidyasagar University 1999………………………………….M.Sc. in Botany, Vidyasagar University 2001-2007…………………………...Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS 1. Mahapatra A., Srinivasan G., Richter K.B., Meyer A., Lienard T., Zhang J.K., Zhao G., Kang P.T., Chan M., Gottschalk G., Metcalf W.W., and J.A. Krzycki (2007) Class I and class II lysyl-tRNA synthetase mutants and the genetic encoding of pyrrolysine in Methanosarcina spp. Mol. Microbiol. 64:1306-18. 2. Longstaff D.G., Larue R.C., Faust J.E., Mahapatra A., Zhang L., Green-church K.B., and J.A. Krzycki. (2007) A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine. Proc. Natl. Acad Sci USA. 104:1021-1026. 3. Mahapatra A., and J.A. Krzycki (2007) The Genetic Code In: McGraw-Hill Yearbook of Science & Technology, McGraw-Hill, New York. 93-98. 4. Mahapatra A., Patel A., Soares J.A., Larue R.C., Zhang J.K., Metcalf W.W., and J.A. Krzycki (2006) Characterization of a Methanosarcina acetivorans mutant unable to translate UAG as pyrrolysine. Mol. Microbiol. 59: 56-66. ix 5. Blight, S. K., Larue, R. C., Mahapatra, A., Longstaff, D. G., Chang, E., Zhao, G., Kang, P. T., Green-Church, K. B., Chan, M. K., and J. A. Krzycki. (2004) Direct charging of tRNACUA with pyrrolysine in vitro and in vivo. Nature. 431: 333- 335. FIELDS OF STUDY Major Field: Microbiology x TABLE OF CONTENTS Page Abstract..................................................................................................................ii Dedication……………………………………………………………………………......v Acknowledgments………………………………………………………………….…...vi Vita…………………………………………………………………………………...…..ix List of Tables…………………………………………………………………………...xv List