Study Of DNA-protein Interaction For GlnR Factor From Streptococcus Agalactiae

In order to research the key amino acid residues with DNA binding sites in GlnRfactor?firstly Predicting DNA binding sites of the GlnR factor of Streptococcus agalactiaeby bioinformatic methods, three critical DNA binding sites of amino acidresidues(R27,R30and R47R48) were screened out in GlnR factors, Then overlapextension PCR was used to mutate thiese three sites separately and construct these glnRmutations and build their prokaryotic expression vector which is efficient expression in E.coli. Finally Purification three target proteins which is research for GlnR-glnRA bindingby the mean of EMSA. Observed three mutated protein and glnRA operon affinity changes,and determined GlnR-DNA three-dimensional structure model, thus preliminaryelucidated GlnR and the mechanism of the DNA.According to the content of the above this paper experimented with the following:1. Forecast Strepococcus agalactiae GlnR factor which probably involved in DNA incombination of amino acid residues through the network services Based on BindN. Thenscreened the most likely binding sites preliminarily by analysis the amino acid residues.Finally, used the web server to predict GlnR-DNA three-dimensional structure model toscreen DNA binding sites. It found the prediction submitted to Zhang Lab network serverpresents two different GlnR-DNA three-dimensional structure model. In order todetermine its structure model, we screened R27(27arginine), R30(30arginine) andR47R48(47and48arginine) as thr possible binding sites and the experimental mutatedsites.2. Mutated three amino acid residues by using overlapping PCR and constructedthree mutated glnR genes. According to its position in the amino acid sequence namedglnRm27, glnRm30and glnRm4748(expressed in glnRm below) respectively. DNAsequencing results showed that R27mutated to A27(GCG to AGA), R30mutated to A30(GCG to CGC), R47R48mutated to A47A48(CGCCGC to GCGCGC).3. Used the fusion expression vector to build pGEX-4t-glnRm27, pGEX-4t-glnRm30,pGEX-4t-glnRm4748successfully. These vectorer were converted to Escherichia coli BL21and optimized IPTG concentration and inducing temperature. The results showed that theoptimal concentration of IPTG inducing expression tendency for0.1mM and the best induction temperature was30?. In addition the soluble tests revealed expression proteinsare soluble. Last we used affinity chromatography column purification.4. The above three kinds of mutant proteins interact with glnRA gene in vitro werestudied respectively by Electrophoretic Mobility Shift Assay (EMSA). By comparing tothe bands which is the result of GlnR protein and glnRA gene interaction, it found thatGlnRm4748protein almost lost the affinity with glnRA completely. While GlnRm27andGlnRm30still is combining with glnRA, but the affinity far weaker than GlnR protein.The conclusion is that1)47,48amino acid residues play a key role on the binding glnRAand GlnR and30amino acid residues did worse.2) the influence of the27also play a roleon GlnR binding glnRA, but its effect on DNA binding relatively weaker;3) it is lesspossibily to formate the hydrogen bonds in Predicted GlnR-DNA three-dimensionalstructure model, the interaction between GlnR and DNA model maybe is Helix-turn-helix(HTH).