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 ^ew riter fiice, while others may be from any type o f 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 afifect 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 comer 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 o f 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. UMI A Bell & Howell Information C om p aiy 300 North Zed) Road, Ann Aibor MI 48106-1346 USA 313/761-4700 800/521-0600 IN VIVO CHARACTERIZATION OF ARCHAEAL TRANSCRIPTION TERMINATION SIGNALS AND CHARACTERIZATION OF A HALOFERAX VOLCANII HEAT SHOCK GENE: A MODEL FOR GENE REGULATION 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 Yen-Ping Kuo, B.S., M.S. ***** The Ohio State University 1997 Dissertation Committee: Approved by Charles J. Daniels, Advisor Tina Henkin John N. Reeve F. Robert Tabita Adviser Department of Microbiology UMI Number: 9731658 UMI Microform 9731658 Copyright 1997, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT To investigate the mechanisms of gene expression in the Archaea we developed an in vivo assay system to examine the requirements for transcription termination in Haloferax volcanii and began studies to identify a model for studying gene regulation. The availability of a transformation system and gene expression vectors for H. volcanii has facilitated the development of a plasmid-based in vivo assay for transcription termination. Using this system, we established that a eukaryal RNA polymerase III terminator was recognized by the archaeal RNA transcription apparatus and functioned as an efficient termination element. This element was combined with the H. volcanii tRNALys promoter and the yeast tRNAProM reporter gene to construct a transcription termination assay module that could be used to investigate the sequence and structural characteristics of archaeal transcription termination signals. Results of this study established that oligo-dT tracts function as efficient terminators in vivo and that a second, bacterial-like rho-independent, termination mechanism may be also present. Furthermore, the termination properties of elements containing two T-tracts suggested the occurrence of an “inch-worming” mechanism for archaeal transcription termination. 11 To study regulated gene expression in H. volcanii, we chose to examine the heat shock response as a potential model system. Using information available for global expression of heat shock loci, we identified and cloned a gene encoding the chaperonin- containing TCP-1 (CCTl) protein. The deduced amino acid sequence of this gene predicted a protein of 59 kDa that was similar to Sulfolobus shibatae TF55P and human TCP-1. Transcripts of cctl and its related genes were present at higher levels when cells were challenged with heat stress or salt shock. Transcript analysis indicated that transcription of the cctl gene initiated 25 bp downstream from a typical archaeal TATA promoter element under both control and heat shock conditions, and analysis of the termination site indicated that the transcript terminated in a T6 T-tract. These studies established the cctl gene as a suitable gene for the study of transcription regulation in H. volcanii. Ill This work is dedicated to my children, Shin and Joey, and my husband, Tenfu, who have often shared family time with my research. IV ACKNOWLEDGMENTS I would like to especially thank my advisor. Dr. Charles J. Daniels, for his guidance, support, understanding and patience over the past five years and in the preparation of this dissertation. I also thank my Committee members. Dr. John Reeve, Dr. F. Robert Tabita, and Dr. Tina Henkin, for their encouragement and advice. 1 am indebted to David Armbruster who has been a wonderful mentor and friend since I joined the laboratory. I am grateful to Dorothea Thompson both for her friendship and her assistance in editing this dissertation. Many thanks also go to my co-workers in Daniels’ lab for creating a fun working environment and introducing me to the heart of American culture. I will be always grateful to my parents who emphasized the value of higher education and gave me unlimited support during my pursuit of a Ph.D. Finally, my sincere appreciation goes to my loving husband for his sacrifice, encouragement and unconditional support. VITA July 18,1962 ............................................Bom—Chia-I, Taiwan 1984 ........................................................B.S., National Taiwan University, Taipei, Taiwan 1984-1987 ........................................... Research Assistant and Medical Technologist, National Taiwan University Hospital, Taipei, Taiwan 1989 ....................................................... M.S., Michigan State University East Lansing, Michigan 1990-1991 ........................................... Microbiologist, Ohio Department of Health Columbus, Ohio 1991 -present ........................................... Graduate Teaching andResearch Associate, The Ohio State University Columbus, Ohio PUBLICATIONS Research Publication Kuo, Y.-P. 1989. Detection of Enterohemorragic Escherichia coli 0157:H7 by radioactive and non-radioactive DNA probes. M.S. Thesis. Michigan State University, East Lansing, MI. VI Abstract Palmer, J. R., Y.-P. Kuo, and C. J. Daniels. 1994. /n v/vo analysis of archaeal transcription signals. American Society for Microbiology, May. Kuo, Y.-P. and M.-C. Shen. 1986. Immunological status of patients with hemophilia in Taiwan. XVII International Congress of the World Federation of Hemophilia, Milano. Kuo, Y. P., C.-H. Wang, and M.-C. Shen. 1986. A test of platelet bound immunoglobulin by electroimmunoassay in thrombocytopenic purpura. Chinese Hematology Association, Taipei. Kuo, Y.-P., F.-W. Liu, R.-F. Hsiu, and M.-C. Shen. 1986. Immunological defects in Hemophiliacs with or without HTLV-III infections. Chinese Hematology Association, Taipei. Chang, S.-C., S. C. Lue, Y.-P. Kuo, and M.-C. Shen. 1986. Analysis of DNA in Hemophilia A patients using a cloned probe. Chinese Hematology Association, Taipei. FIELD OF STUDY Major Field: Microbiology Molecular Biology of the Archaea vu TABLE OF CONTENTS ABSTRACT.......................................................................................................................... ii DEDICATION..................................................................................................................... iv ACKNOWLEDGMENTS....................................................................................................v VITA.....................................................................................................................................vi TABLE OF CONTENTS.................................................................................................. viii LIST OF FIGURES...........................................................................................................xiii LIST OF TABLES.............................................................................................................xvi LIST OF ABBREVIATIONS.......................................................................................... xvii CHAPTER 1: GENERAL INTRODUCTION....................................................................I Archaea and the Three Domains of Life ...........................................................................1 Gene Transcription ............................................................................................................6 DNA-dependent RNA polymerases ............................................................................. 6 Promoter, Transcription Initiation Factors, and Initiation Complex Formation 13 Transcription Elongation ............................................................................................17 Transcription Termination ..........................................................................................19 Heat Shock Proteins: Structure and Function ................................................................ 25 HSP70 family ..............................................................................................................26 Hsp60 family ...............................................................................................................28 CCT/TriC/TCP-l/TF55 Family