A Stmcture/Function Analysis of the Interaction of the E.coli NusA protein with RNA polymerase, the phage h N protein, and nut site RNA Thien-Fah Mah A thesis submitted in canformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular and Medical Genetics University of Toronto O Copyright by Thien-Fah Mah 1999 National Library Bibliothèque nationale 1*1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wewymn OnawaON KIAW OaawôoN KlAW Canada CaMda YaoY vai. rowwnco Our W Nari. 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A Structure/Function Analysis of the Interaction of the E.coli NusA protein with RNA polymerase, the phage h N pmtein. and nut site RNA by Thien-Fah Mah Department of MolecuIar and Medicai Genetics in the University of Toronto Submitted for a Doctor of Philosophy degree, 1999 Abstract NusA is an E-coli protein that controls transcription elongation. terminrition and mtitermination. In this thesis. 1 show that the functions of NusA in transcription are fxilitated by interactions with RNA polymerase, RNA and the h N protein. Use of a series of deletion constructs of NusA ailowed me to identify specific regions of NusA involved in specific interactions and in various aspects of NusA function. Genetic evidence suggested that NusA may interact with the box4 portion of the N-utilization site (nut site = ho-rA, interbox, and boxB). By constructing multiple nucleotide siibstitutions in the nut site, 1 showed that the identities of certain nucleotides lit the 3' end of bosA and in the interbox were important for NusA to associatc with an N- utlr site cornplex. NusA association with RNA in the presence of N is presurnably fxilitatcd by its S I and KH homology regions, two types of RNA-binding domains in NusA. Elimination or mutation of the S 1 homology region prevented the association of XusA ~vithan N-ntrt site cornplex. Using affinity chromatognphy experirnents. 1 found that RNA polymense bound equally well to an amino-terminal RNA polymerase-binding region in amino acids 1-137 and a carboxy-terminal RNA polymerase-binding region in arnino acids 232495 of NusA. By contrast. the a subunit of RNA polymerase only bound to the carboxy- terminal RNA polymerase-binding region of NusA. N protein also bound to ri carboxy- terminal region of NusA. and both N and a allowed NusA to associate with RNA in a gel mobility shift assay. When the carboxy-terminal region of NusA was deleted in NusA ( 1-348). the loss of N-binding and a-binding ability did not abolish NusA function in termination and antitemination assays. This minimal functional NusA protein retained the KH and S 1 homology regions and the amino-terminal RNA polymerase-binding rcsion. Unlike full length NusA (1-495). NusA (1-416) could bind RNA on its own. These observations suggest thclt the carboxy-terminal region of NusA inhibits RiVA binding and that this inhibition cmbe relieved by interaction with the h N protrin or the cc subunit of RNA polymerase. Table of Contents Thesis Abstract ii Table of Contents iv Ac knowledgements vii List of Figures viii List of Tables X Chapter 1: Intrduction 11 Transcription in E.co/i 11 Initiation 12 Elongation 15 Termination 23 Rho-independent temination 24 Rho dependent termination 26 Regulation of Elongation by the Bacteriophage h N protein 28 The nrrt site 30 The Processive Antitermination Cornpiex 33 N protein 33 Nus factors 35 Mechanism of Antiterrnination 45 Other Antitermination Systems 46 Q-mediated Antitermination 46 rrn Antitermination 48 pr~rand nun in phage HK022 50 Thesis Ra tionale 52 Chapter 2: Functional Importance of Regions in E.coli Elongation Factor NusA that Interact with RNA polymerase, the Bacteriophage h N protein, and RNA 53 Abstract Introduction Results Discussion Expcrimental Procedures Chapter 3: The a Subunit of Exoli RNA pdymerase Activates RNA-Binding by NusA Abstract Introduction Rcsults Discussion Esperimental Procedures Chapter 4: Interaction Between the E-colr' NusA Elongation Factor and the h mrt site RNA Abstract Introduction Rcsults Discussion Espcrimental Procedures Chapter 5: Thesis summary and Future Directions References Acknowledgements I have learned an immeasurable arnount about science from my supervisor, Jack Greenblatt. 1 started in the lab with very little expenence. Through his signature style of supervision. 1 have grown into a scientist who feels cornfortable defending her results and arguing points with peers. 1 would like to thank the many people who have ken in the GreenbIatt lab who have filied the years with laughter, support, advice and good mernories. As fellow collaborators on the prokaryotic project. Joyce Li and Jeremy Mogridge provided vaiuable reagents and invduable advice. My committee members. Brenda Andrews, Rick Collins and Barbara Funnell, have been absolutely wonderful. 1 couid not have asked for more caring and comrnitted individuals to help guide me through graduate school. This degree has taken a long time to complete. It would have ken easy to get lost along the \vay. but 1 have ken fortunate to have met many arnazing people. This has included people both in the Department. as well as ones who are out working in the wild worId. who kept me grounded with their friendship. iMy Family has ken there al1 the way. Thanks for believing in me. Last. but not least. 1 would like to thank Raja Bhattacharyya for unwavering love and support. vii List of Figures Chapter 1 The Structural Features of the Elongation Complex The Regulatory eiements of the h genome The h. 11rlrR site The Comple te N-modi fied Antitermination Complex Chapter 2 Circular dichroism analysis of NusA deletion constructs 58 NMR analysis of fragments of NusA 60 Two RNA poiymerase-binding regions in NusA 62 Rcgions of NusA important for enhancement of termination irl vitro 64 N binds a carboxy-terminal region of NusA 66 Region3 of' NusA necessary for enhancement of antitermination by N 68 The effects of NusA deletions of the formation of N-NusA-riicr site complexes 71 The effects of NusA deletions on the formation of cornpletc complexes contüining N. the NusA factors and RNA polymerase 73 Summary of the Results of the Deletion Andysis of NusA 77 Chapter 3 Binding of NusA to RNA in the presence of the RNA polymerase a subunit 89 a-CTD stimultes RNA-binding by NusA 91 RNA-binding by NusA in the presence of a is sequence-specific and sensitive to a mutation in the S 1 domain of NusA Interaction of oc with the carboxy-terminal region of NusA ... Vlll A carboxy-terrninally truncated NusA binds specifically to rzrrt site RNA 97 The À N protein binds directly to the carboxy-terminal 193 amino acids of NusA 99 Modcl for N USAfunction in elongation, termination and antitermination 101 Chapter 4 Substitution of nucleotides 1 to 3 in box4 does not affect the ability of NusA ( 1-416) to interact with rzrrf site RNA, whereas substitution of nucleotides 3 to 6 or nuclsotides 7 to 9 substantially reduces the interaction 114 Substit~itionof nucleotides 10 to 12 of ho.rA and 13 to 17 of the interbox both ssverely affect the ability of NusA ( 1-4 16) to interact with nrir site RNA 115 Substitution of nucleotide 23 in the loop of hoxB reduces the ability of NusA ( 1-316) to interact with nrrr site RNA whereas substitution of nucleotide 31 has li tt le effect 117 Substit~itionof nucleotide 27 in the loop of boxB severely reduces the ability of NiisA ( 1-4 16) to interact with rzrtt site RNA whereas substitution of nucleotides 25 and 26 has little effect 118 Mutations in the S 1 homology region of NusA have differential effects on the formation o t' N-NusA-nitr site complexes 121 Mutations in the S 1 homology region of NusA have differential effects on the formation of ri complete complex containing RNA polymerase. N. NusA. NusB. NrisG and S 10 List of Tables Chapter 4 Comparison of the abilities of NusA ( 1416). in the absence of N. and wild type NusA. in tIic presence of S. to bind to wild type and mutant nrit sites 119 Introduction In this thesis, 1 describe experiments performed to elucidate the role of the E.~clzer-icltilrcoli NusA protein in the control of transcription during elongation and termination as well as during transcriptional antiterrnination medirtted by the bacteriophage h X protei n. Therefore, 1 kgin with a detailed description of transcription by E-coliRNA pol y merasc. Transcrintian in EcoIi Transcription in Esclwrichia coli is carried out by a single multi-subunit RNA po1yrncr;isc. This process cün be separated into three distinct stages: initiation. elongation and tcrmination. Initiation is characterized by RNA polymerase binding to promoter DNA. conversion ot'an unstably bound RNA polymerase to a stably bound one.