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Title: Exploring inhibition of the bacterial enhancer binding protein PspF and transcription factor sigma 54 using peptide-based chemical genetic approaches
Author: Rayner, Lucy Jane
ISNI:       0000 0004 2735 4722
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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AAA+ proteins (ATPases Associated with various cellular Activities) are a sub-family of P-loop NTPases which convert chemical energy into physical motion to carry out a multitude of functions. The AAA+ protein phage shock protein F (PspF) is a bacterial enhancer binding protein (bEBP) for the major variant transcription factor sigma 54 (σ54) in E. coli. PspF binds to and remodels the promoter DNA bound σ54-RNA polymerase holoenzyme (Eσ54), inducing isomerisation from a transcriptionally silent closed complex to an open complex. Without binding to and concurrent ATP hydrolysis by the bEBP, Eσ54 is unable to spontaneously form the transcriptionally competent open complex, thus maintaining tight control over its associated genes. Conformational changes in the PspF ATP binding site upon hydrolysis are communicated through a pathway of conformational changes in PspF, resulting in the reorganisation of two mobile loops – termed Loop 1 and Loop 2. Loop 1 contains the GAFTGA motif, highly conserved across bEBPs, which forms a stable interaction with σ54. Thus far, traditional approaches such as site-directed mutagenesis and cryo-EM have been used to probe the conformational changes produced at each stage of nucleotide hydrolysis. In this work, the PspF/ σ54 interaction is investigated using a chemical genetic approach. The interactions of designed synthetic peptides with σ54 are investigated using in vitro techniques including a dansyl-based fluorescence binding assay, isothermal titration calorimetry (ITC) and traditional activity based transcription assays. Maltose binding protein fusions of the PspF fragments carrying Loop 1 were also investigated using an in vivo β-galactosidase assay. Finally, Split-Intein Mediated Circular Ligation of Peptides and Proteins (SICLOPPS) was used to create two plasmid-borne cyclic peptide libraries, one random and the other containing the GAFTGA motif. To select for peptides inhibitory to the PspF-σ54 interaction, a selection system was developed based on a promoter-lacZ fusion and the conditionally toxic lactose analogue phenyl-β-D-galactose (Pgal). To confirm hits, a secondary screen based on a structurally and functionally related σ54-bEBP interaction was developed and several peptides active in vivo were identified.
Supervisor: Tate, Ed ; Buck, Martin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral