INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Beil & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 9219007 Transfer RNA maturation in the archaebacterium Haloferax volcanii: A study of ribonuclease P and transfer RNA intron endonuclease Nieuwlandt, Daniel Terry, Ph.D. The Ohio State University, 1992 UMI 300 N. ZeebRd. Ann Arbor, MI 48106 TRANSFER RNA MATURATION IN THE ARCHAEBACTERIUM HALOFERAX VOLCANII: A STUDY OF RIBONUCLEASE P AND TRANSFER RNA INTRON ENDONUCLEASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Daniel Terry Nieuwlandt, M.S. 3|« * £ % Ht The Ohio State University 1992 Dissertation Committee: Approved by Dr. C. Daniels Dr. A. Darzins Dr. J. Krzycki Advisor Dr. J. Reeve Department of Microbiology To Mom and Dad ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. Chuck Daniels for his guidance and patience throughout this project. I would also like to express my appreciation for the time and effort provided by Drs. A1 Darzins, Darrell Galloway, Joe Krzycki, and John Reeve as members of my Dissertation committee. Thanks go to the members of the John Reeve and Joe Krzycki labs for their friendship and many helpful discussions. Special thanks go to Dr. Leo Thompson, my "mentor", for his help and friendship, and to Dr. Elizabeth Haas and Dr. Jim Brown for their advice and contributions to the RNase P research. Jorge Acevado, Dave Armbruster, Bill Oda, Traci Baltrus, Linda McCart, and Mary Beth Carr also deserve special thanks for their help and for making our lab an interesting and fun place to work. VITA February 5, 1962 Bom - Whittier, California 1985 .................. B. A., California State University, Fullerton, California 1988 M. S., Iowa State University, Ames, Iowa 1988-Present Graduate Research Associate, Department of Microbiology, The Ohio State University PUBLICATIONS Nieuwlandt, D. T., E. S. Haas, and C. J. Daniels. 1991. The RNA component of RNase P from the archaebacterium Haloferax volcanii. J. Biol. Chem. 266: 5689-5695. Nieuwlandt, D. T., and C. J. Daniels. 1990. An expression vector for the archaebacterium Haloferax volcanii. J. Bacteriol. 172:7104-7110. Thompson, L. D., L. D. Brandon, D. T. Nieuwlandt, and C. J. Daniels. 1989. Transfer RNA intron processing in the halophilic archaebacteria. Can. J. Microbiol. 35: 36-42. Nieuwlandt, D. T., and P. A. Pattee. 1989. Transformation of a conditional peptidoglycan-deficient mutant of Staphylococcus aureus with plasmid DNA. J. Bacteriol. 171: 4906-4913. Nieuwlandt, D. T. 1988. Genetic analysis and plasmid transformation of a temperature-sensitive osmotically fragile mutant of Staphylococcus aureus. M. S. Thesis. Iowa State University, Ames, LA. FIELD OF STUDY Major Field: Microbiology Studies in Archaebacterial Molecular Biology I v TABLE OF CONTENTS DEDICATION ......................................................................................................................... ii ACKNOWLEDGMENTS ................................................................................................ iii VITA ...................................................................................................................................... iv LIST OF TABLES .............................................................................................. 7 ............ viii LIST OF FIGURES............................................................................................................... ix LIST OF ABBREVIATIONS.............................................................................................. xi PAGE INTRODUCTION ................................................................................................................. 1 LITERATURE REVIEW ...................................................................................................... 5 Archaebacteria........................................................................................................... 6 Overview of the archaebacteria ................................................................. 6 Gene structure and expression in the archaebacteria ...............................18 Archaebacterial plasmids and phages ...................................................... 25 Genetic exchange systems in the archaebacteria.....................................28 Ribonuclease P ............................................................................................................39 Overview of ribonuclease P .................................................................... 39 Discovery of RNase P ................................................................................. 41 Physical properties of RNase P ..................................................................42 Properties of the RNase P reaction ......................................................... 46 Structure of RNase P R N A .........................................................................50 RNase P RNA structure-function relationships ..................................... 58 Substrate recognition by RNase P .............................................................. 62 tRNA intron endonuclease .................................................................................... 67 Overview of intron processing .................................................................... 67 Nuclear pre-tRNA introns ...........................................................................73 Archaebacterial pre-tRNA introns ...............................................................84 Archaebacterial pre-rRNA introns .............................................................. 90 MATERIALS AND METHODS ......................................................................................... 92 Bacterial strains and plasm ids .............................................................................. 93 Small scale isolation of plasmid DNA from E. coH and H. volcanii ...................94 Large scale isolation of plasmid DNA from E. coH ........................................ 95 Large scale isolation of plasmid DNA from H. volcanii ................................. 96 Isolation of H. volcanii chromosomal DNA ...................................................... 97 V Isolation of double-stranded replicative form (RF) bacteriophage M 1 3 D N A ....................................................................................................... 97 Isolation of bacteriophage M13 single-stranded DN A ......................................... 98 Isolation of H. volcanii and T. acidophilum R N A ........................................ 99 Oligonucleotide preparation ...................................................................................... 99 Determination of RNA, DNA, and protein concentrations ............................. 100 Nucleic acid precipitation ........................................................................................ 100 Electrophoresis and visualization of nucleic acids and proteins ..................... 101 U.V. shadowing of nucleic acids ....................................................................... 105 Recovery of RNA and DNA from electrophoresis gels .................................. 105 Restriction and ligation of D N A ......................................................................... 106 In vitro transcription reactions ............................................................................ 107 Radiolabeling of RNA and DNA ............................................................................108 Preparation of transformation- and transfection-competent E. coU ................ 110 Transformation and transfection of E. coli .........................................................I l l H. volcanii expression vector construction ...................................................... 112 Shuttle-expression vector copy number determination .......................................113 Reconstruction of Haloferax tRNATrP genes using the polymerase chain reaction .................................................................................................