Protein Binding and Crowding Effects on DNA Condensation
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- Institutt for fysikk 
The main goal of this work is to map how bacterial nucleoid associated proteins (NAPs)condense DNA in tandem with a crowded environment, since this may provide usefulinformation for research where DNA compaction and protein binding is of high importance,such as protein expression, gene therapy and cancer research. The isolated effectsof NAPs on condensation are subject to constant investigation, but little is known aboutthe combination of both protein binding and crowding effects in vitro. The combinedeffects of crowding and condensing agents on DNA compaction are investigated with fluorescencespectroscopy (FS), fluorescence correlation spectroscopy (FCS), atomic forcemicroscopy (AFM), cell-free expression assays and electrophoresis mobility shift assays(EMSAs). Plasmid pSB-M2g-1-17 was harvested from DH5-α Escherichia coli, and subjectedto polyethylene glycol (PEG) and the condensing polyamine spermine. Histone-likenucleoid-structuring protein (H-NS) was over-expressed, purified and its activity confirmed. A 4017 bp amplicon from template plasmid pSB-E1g, including a T7 promoter anda gene encoding green fluorescent protein (GFP), was used to quantify DNA compactionand protein expression under influence of PEG and H-NS. The combined effect of PEGand spermine was found to reduce the hydrodynamic volume of DNA to 5% of its initialvalue, while spermine and PEG alone reduced it to 33% and 23%, respectively, althoughwith considerable standard deviations. 0.5 to 1.0 µM H-NS was found sufficient to inhibitcell-free expression of GFP, and 5 µM induced visual DNA-aggregation by AFM. Novelinsight in synergism was attained, as crowding was shown to greatly reduce the amount ofH-NS required for condensation as measured by FCS.