The E. coli DnaA protein initiates chromosome replication and regulates gene transcription by binding to DnaA boxes, the double-strand nonamers with the consensus sequence TTA/TTNCACA. I intended to investigate the interactions between DnaA protein and DNA by Molecular biology and NMR methods. DnaA domain IV which contains the Helix-turn-Helix (HTH) motif is used as a functional model for studying DnaA box binding in vitro. I purified C-terminal domain IV (C98 aa 370-467) of different mutant DnaA proteins by GST method to investigate the binding sites and binding preference for oligonucleotides variants carrying DnaA boxes. The six mutations (R407K, H434R, H434K, P423S, P423I and P423R) I selected are located in evolutionarily conserved residues which interact directly with DNA. Gel shift DNA binding assays with DnaAc98 mutant proteins showed that H434R and H434K strongly decreased the binding affinity, while R407K did not affect affinity as much as I expected from the in vivo complementation assay, indicating that the inefficiency was due to protein instability. P423I was wt alike, P423S, and P423R generally kept high binding activity. The λ Red recombination method was used to introduce the P423I mutation into dnaA in the E.coli chromosome. The effect of P423I on in vivo binding to DnaA boxes was determined by measuring expression from two DnaA regulated promoters (pdnaA and pmioC) using single copy lacZ fusions, and by studying replication by Flow cytometry. P423I increased expression of the two promoters by 20% and showed moderate initiation asynchrony in Flow cytometry assay. The results suggested that P423I had an overall deficiency in box binding and initiation regulation which might due to its preference for weak boxes. I was going to use NMR method to investigate interactions and confirm changes of binding features between mutant DnaAc98 proteins and DNA which were observed in biological experiments. Because of various objective reasons, I only studied wild type DnaA domain IV without DnaA box in this report as the background for further researches. The 1D 1H NMR and 2D NOESY and TOCSY were performed on 600MHz and 15N labeled sample for 2D 15N-HSQC and 3D 15N-HSQC NOESY/TOCSY experiments were measured on 800MHz. However, 1D 1H NMR and 2D TOCSY spectra indicated that some DNA impurities left in purified protein sample which might be because the DNA fragment was protected by DnaA binding at Dnase I digestion step during the purification. The results of 15N labeled sample were compared with previous published data and showed that some residues that had significant chemical shifts change in 15N-HSQC spectrum, which might be involved in the DNA binding.
|Uddannelser||Molekylærbiologi, (Bachelor/kandidatuddannelse) KandidatKemi, (Bachelor/kandidatuddannelse) Kandidat|
|Udgivelsesdato||25 sep. 2008|
|Vejledere||Tove Atlung & Poul Erik Hansen|
- DnaA box
- gel shift assay
- Red recombination