Sunday, November 11, 2007

More results from Sunita and Andrew

Our results that suggest Sxy binds to DNA are exciting but suspicious; all Sxy-DNA binding data can be explained by the presence of contaminating CRP in the Sxy protein preps. This is because the Sxy-DNA binding data is identical to CRP-DNA data. First, EcSxy binds DNA but HiSxy does not. Second, EcSxy greatly prefers the pilA-N (CRP-N mutant) site over the wildtype pilA CRP-S site. Third, when EcSxy and EcCRP are mixed together, only one protein binds to a DNA molecule, suggesting that both proteins target the same site (this is consistent with the pilA-N data).

The next set of experiments is clear: 1) Test whether EcSxy can bind DNA in the absence of cAMP (EcCRP cannot), 2) Test whether EcSxy binds to a pilA promoter that lacks its CRP site, 3) Use western blots to probe for EcCRP in the Sxy preps, and 4) Test DNA binding by EcSxy that has been isolated from a crp- expression strain.

However, several arguments can still be made that EcSxy does in fact bind DNA. First, EcSxy and HiSxy were isolated form the same E. coli strain using the same procedure, thus we would expect EcCRP to contaminate the HiSxy preps as well (which clearly has not happened because HiSxy preps don’t bind DNA). Second, far-western analysis has not detected EcCRP in the Sxy preps.

If tomorrow’s experiments show that EcSxy binds DNA in the absence of cAMP, two new hypotheses need to be addressed: 1) Does EcSxy prefer the pilA-N promoter not because it binds the CRP-N site, but because the CRP-N site makes DNA more bendable than the wildtype CRP-S promoter? 2) Does HiSxy fail to bind DNA because bandshift reaction conditions are not favourable for H. influenzae proteins?


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