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Functional Analysis of Deletion Mutants of Fino, the Fertility Inhibition Protein of the F-Like Plasmids

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Functional Analysis of Deletion Mutants of Fino, the Fertility Inhibition Protein of the F-Like Plasmids University of Alberta Functional Analysis of Deletion Mutants of FinO, the Fertility Inhibition Protein of the F-like Plasmids James R. Sandercock O A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillrnent of the requirements for the degree of .MASTER OF SCEXCE. DEPARTMENT OF BIOLOGICAL SCIENCES Edmonton. Aiberta Spnng, 1998 National Library Bibliothéque nationale I*m of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. nie Wellington Ottawa ON Kt A ON4 CMawaON KIA ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence alIowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in rnicroform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/^, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. To my parents. Bob and Libby, who's love and support are unending. CI To my sister. Knansie, who (incessantly) challenges my world-view. Abstract High frequency conjugative transfer of the F-like piasmids is possible when TraJ protein. the main activator of uyiscriprion of the transfer operon. is synthesized in the donor organism. Transfer is negatively regulated by the FinP antisense RNA. which hybridizes to the positive sense transcript. Td. thereby blocking TraJ protein synthesis. The FinO protein inhibits transfer of the plasmid by protecting FinP RNA from degradation. and by promoting duplex formation between the FinP and Trd transcripts. The purpose of this thesis is to delineare the minimal sequence required for fenility inhibition. as mediated by FinO. and to define the regions of the protein that are responsible for the above functions. The fi0 gene and downstrearn sequences from plasmid R6-5 were cloned into the multicopy plasrnid pBC-KS+. A series of nested deletions of the downstrearn sequences were created. and the deletion products were subcloned. Clones which contained the deletion products were assayed by a number of means. The results indicate that the downstrem sequences do not play a role in fertility inhibition. and that minor deletions of the 3' end of thefin0 gene result in the loss of fertility inhibition. Frag,omnts of the fino gene were translationally fused to the 3' end of the gst gene of the expression vector pGEX-2T. The gst-fusion constructs were examined for their ability to repress conjugal transfer, and for their ability to stabilize FinP RNA in vivo. GST-fusion peptides were purïfied and assessed for their ability to bind FinP RNA and prornote duplex formation between FinP and TraJ uanscnpts in vitro. The specificity of binding was also examined. GST-Fin0 protein bound FinP RNA with hi& affinity. with an association constant (ka)of 5.6 x 10' Mi.Binding specificity assays indicated that GST-Fin0 bound the target RNA, FhP, with a moderate degree of specificity. The protein increased the rate of duplex formation between the minscripts -3.7-fold in vitro. The plasmid which encodes GST-FtnO. pGEX- F02. reduced mating efficiency -300-fold. and stabilized FinP in L+VO. with a half iife > 60 minutes. The fusion product GST-Fin073. which includes the N- terminal 73 amino acids of FinO. bound FinP with a ka of -2.2 x 10' M-'. and displayed a moderate specificity for the FinP target. GST-Fin073 promoted duplex formation -3-fold in vitro. The peptide conferred no stability to FinP vanscripts when expressed in vivo from the plasmid pGEX-F073. The intermediate sized peptide, GST-Fin0141 bound FinP with a ka of 3.5 x 10' M-'. and displayed a modente specificity for the target RNA. The peptide increased the duplex formation rate 19-fold in vitro. The peptide did not confer iertility inhibition or FinP stability when 1expressed from the plasmid pGEX-F0 14 1 in vivo. From these data a mode1 was developed which proposes a molecular mechanism by which FinO protein protects FinP from degradation. and by which FinO promotes duplex formation between FinP and Trd. thereby repressing expression of the uansfer operon. Acknowledgements Fint and foremost I want to thank my advisor. Dr. Laura Frost. for giving me a place in the lab to hang my hat. Throughout the fint few frustrating years you have been much more than supportive. Thank you for talking me down from the Iedge on more than one occasion. Oh. and thanks for the Scotch. Thdsalso go out to Tirn van Biesen. who preceded me on the Fin0 project. -lot only did he blaze the GST-Fin0 uail. but he also left several constructs. which proved to be invaluable for much of my research. Karen Anthony has been of geat help throughout my stay in the lab. Karen. you make an excellent senior graduate student. and you've contnbuted to my molecular tutelage in many ways. Lori Jerome has been my partner in crime on the FinOP system. She introduced me to the big bad world of RNA. By rny estimation I'd have accomplished very littk without her laboratory expertise and overall level-headedness. Lon. there is not enough thanks by which I can ;ive you proper credit. Big Bill Kiimke has been the "Frost Lab Guru" of wit. sarcasm. and computen for the Iast three years. ï"hanlis Bill for your hefp with GCG, internet stuff. and Civ. 1 also extend thanks to Jan Manchak, Richard Fekete. John Simon and the many summer students/speciaI project students who made my stay in the Frost Lab enjoyable. To my science mentors. Tony Cornish. Brent Rudy and Linda. Thank you for showing me the ropes in my under~nduateyears. You taught me much about science. and it's proper place in one's life. Kudos to Dr. Roy, who (unceremoniously) kicked my hind end into high gear in my third year of undeqpduate studies. Dr. Roy. Tom Hantos and Tony Cornish. you have shown me through word and deed how to be a professional and excellent instructor. Finally, and with considenble fan-fare ... thank you to the Young Life community. Colin. Gilles. Anthony and Kevin. you are the ones who kept me sane throughout my years of graduate snidies. Thank you Gilles for adopting me into the fold! Thanks also to the YL "kids" who's friendships have been some of the most meanin&gfÙl. And Steve ... the thesis would have never flown without pur help with Adobe. Table of Contents Page Chapter 1. Introduction A. Conjugation -3 1. Classification of plasmids 5 II. The F plasrnid 6 B. Regulation of Transfer in F 11 1. TraJ. the main activator of the rra operon 12 II. FinP. an antisense RNA 15 DI. FinO. an RNA binding protein 20 W. interaction of FinO with Fis0 RNA 24 C. Regulation of Trmsfer in RI00 and Rlûû- 1 24 1. R IO0 FinP and TraJ RNA 25 II. TraM and FinM transcripts as anti-antisense RNAs 28 m. The role of FinO in RIO0 29 Ela, FinO as an antisense RNA 29 W. FinO protein as a replator of traM expression 3 1 D. Other Sense-Antisense RNA Systems 32 1. Replication convol of phsrnid ColE 1 33 II. Copy number convol in incm plasmids 37 III. Copy number control in pT 18 1 and piP5O 1 40 N. Control of transposition events of the TnIOlISlO elements E. RNA binding proteins 1- Rom/Rop protein of plasmid CoiE l II. RNA binding and duplex promotion by the protein p53 III. Tat protein and TAR EWA of HTV-1 and HIV-2 N. Rev protein and RRE RNA of HN-1and HIV-2 F. Summary G. Research Objectives Chapter 2. Matenais and Methods Strains and plasmids Chemicals and enzymes Exonuclease III deletions Mating assays P-galactosidase assays Preparation of RNA and nonhem transfers Construction of pGEX-fusion plasmids GST-fusion protein purification In vitro transcriptions ~Mobilityshift assays: RIVA bindin;. competition assays Duplex formation assays Cdculations 1. Cdculation of the association equilibrium constant. ka II. Cdculation of the binding rate constant. k,, Chapter 3. Deletion Mutations of the Ferüüty Inhibition Protein FinO Cause Derepression of Transfer of the F plasmid A. Introduction 75 B. Results 76 1. Exonuclease digestion offin0 region of R6.5 76 II. Qualitative assays of Fin0 activity in vivo 8 1 m. Mating inhibition by R6.5finO deletion derivatives 83 C. Discussion 84 Chapter 1. Characterization of the Functional Domains of FînO, the Fertüïty Inhibition Protein of F-üke Piasmids. A. Introduction 87 B. Results 87 1. Creation of truncated GST-Fi0 constructs 87 II. Conjugation is not inhibited by uuncated GST-Fin0 products 9 1 III. GST-Fin0 derivatives do not stabilize FinP RNA in vivo 95 W. The N-terminal a-helical domain is required for FinP binding 98 V. FinP RNA binding by GST-Fin0 derivatives is specific 1 10 VI. Duplex formation rate is improved by GST-Fin0 derivatives 115 C- Discussion 126 Chapter 5. Discussion A. The role of downstrearn sequences in fenility inhibition 134 B. RNA binding mtivity of Fin0 135 C. RNA protection activity of Fin0 136 D. Promotion of duplex formation by Fin0 137 E. A rnodel of the functionai domains of Fin0 139 F.
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