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Nucleosomes, Transcription and Transcription Regulation in Archaea

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Nucleosomes, Transcription and Transcription Regulation in Archaea NUCLEOSOMES, TRANSCRIPTION AND TRANSCRIPTION REGULATION IN ARCHAEA DISSERTATION Presented in Partial Fulfillment of the requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Yunwei Xie, B.S. ***** The Ohio State University 2005 Dissertation Committee: approved by Dr. John N. Reeve, Advisor Dr. Tina Henkin ______________________ Dr. Michael Ibba Advisor Dr. Irina Artsimovitch Graduate Program in Microbiology ABSTRACT Archaea form the third domain of life separate from Bacteria and Eukarya (Woese et al., 1990). They are single-celled prokaryotic microorganisms with very diverse physiologies, including methanogenesis, halophily and acidophily. The RNA polymerase (RNAP) and the basal transcription machinery in Archaea are more closely related to their eukaryal than bacterial counterparts (Lange et al., 1995; Bell and Jackson, 2001). Many Archaea also contain archaeal histones, which are sequence and structural homologs of eukaryal nucleosomal core histones (Reeve, 2003). I have taken advantage of an in vitro transcription system derived from Methanothermobacter thermautotrophicus (M.t.) (Darcy et al., 1999) to investigate several aspects of archaeal transcription, from initiation to elongation to promoter-specific regulation. Several interesting and significant results were obtained. First, the archaeal RNAP is able to transcribe through one or two archaeal nucleosomes in vitro, albeit at a slower rate. Second, archaeal TATA-binding protein (TBP) remains bound to promoter DNA, but archaeal transcription factor B (TFB) is released from promoter DNA after transcription initiation. Third, transcription of the trpEGCFBAD operon in M.t. is under the regulation of a tryptophan-sensing repressor protein, TRPY. The binding sites for TRPY were identified and the molecular basis for TRPY regulation of the trpEGCFBAD operon expression established. ii ACKNOWLEDGEMENTS I am most grateful to Dr. John N. Reeve as my mentor in the past six years. Dr. Reeve is not only an exemplary advisor, but also a trusted friend. I also want to thank Dr. Reeve and my thesis committee members for carefully reading and correcting this thesis. In addition, I would like to thank all members of the Reeve lab for their teaching and sharing. iii VITA 1977……………………………………………………………………….Born in China 1998……………………………………….B.S. in Biochemistry, Zhongshan University 1999-2005……………………………………...Graduate student, Ohio State University PUBLICATIONS Xie, Y. and J. N. Reeve (2003). In vitro transcription assays using components from Methanothermobacter thermautotrophicus. Methods Enzymol 370: 66-72. Xie, Y. and J. N. Reeve (2004). Transcription by an archaeal RNA polymerase is slowed but not blocked by an archaeal nucleosome. J Bacteriol 186(11): 3492-3498. Xie, Y. and J. N. Reeve (2004). Transcription by Methanothermobacter thermautotrophicus RNA polymerase in vitro releases archaeal transcription factor B but not TATA-box binding protein from the template DNA. J Bacteriol 186(18): 6306-6310. Xie, Y. and J.N. Reeve (2005). Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus. J Bacteriol 187(18): 6419-6429. FIELD OF STUDY Major Field: Microbiology Minor Field: Molecular Biology iv TABLE OF CONTENTS Abstract............................................................................................................................... ii Acknowledgements............................................................................................................ iii Vita..................................................................................................................................... iv List of figures.......................................................................................................................x List of abbreviations ......................................................................................................... xii Chapters: 1. General introduction ......................................................................................................1 Eukaryal histones and nucleosomes.........................................................................2 Archaeal histones and nucleosomes.........................................................................5 Eukaryal basal transcription machinery.................................................................10 Chromatin and transcription in Eukarya................................................................16 Archaeal basal transcription machinery.................................................................17 Archaeal chromatin and transcription....................................................................22 Transcription regulation in Archaea ......................................................................24 2. Transcription by an archaeal RNAP is slowed but not blocked by archaeal nucleosomes...................................................................................................28 Introduction............................................................................................................28 Materials and Methods...........................................................................................30 v Chemicals and reagents.................................................................................30 Construction of transcription templates........................................................30 Growth of M.t................................................................................................30 Purification of M.t. RNAP ............................................................................32 Promoter-independent transcription assay....................................................33 Purification of M.t. TBP and TFB ................................................................34 Purification of archaeal histone HMtA2.......................................................35 Agarose gel shift assay..................................................................................36 [32P]-labeling of DNA templates ..................................................................36 Polyacrylamide gel shift assay......................................................................36 Micrococcal nuclease and primer extension footprinting.............................37 In vitro transcription .....................................................................................38 Ternary complex isolation and stalled transcript elongation ........................38 Results....................................................................................................................39 Construction of T284 that positions a single nucleosome ............................39 Positioning of HMtA2 on T284 ....................................................................40 Polyacrylamide gel shift assay of HMtA2 to T284 ......................................41 In vitro transcription of nucleosomal templates............................................41 HMtA2 assembly did not prevent transcript elongation...............................42 HMtA2 binding decreased the rate of elongation.........................................43 Construction of T435 that positions two nucleosomes.................................43 Transcription through two nucleosomes.......................................................44 Discussion..............................................................................................................45 vi Assembly of HMtA2 on T284 ......................................................................45 Stability of an archaeal elongation complex.................................................48 M.t. RNAP transcription through an archaeal nucleosome...........................49 M.t. RNAP transcription through two archaeal nucleosomes.......................51 Regulation of gene expression by archaeal nucleosome assembly...............51 Potential weakness of the in vitro experiments.............................................53 3. Transcription initiation by an archaeal RNAP in vitro releases TFB, but not TBP from the promoter DNA..........................................................................71 Introduction............................................................................................................71 Materials and Methods...........................................................................................72 Chemicals and reagents.................................................................................72 Template construction...................................................................................72 Single and multiple round transcriptions ......................................................72 Template competition assays ........................................................................73 Immunodetection of TBP and TFB...............................................................74 Results....................................................................................................................75 Multiple-round transcription in vitro ............................................................75 Template competition ...................................................................................76 Immunodetection of TBP and TFB...............................................................77 Discussion..............................................................................................................78 4. In vitro
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