Tuesday, October 23, 2007

Covariance in CRP sites

Yesterday in lab meeting I described how I will introduce base changes at various positions in ICAP to test which positions are most important for HiCRP binding (see previous blog). One important feature of CRP sites, both in E. coli and H. influenzae, is that they have A/T rich runs at either end (positions 1-3 and 20-22; see figure showing a sequence logo from alignment of 45 H. influenzae CRP sites). These A/T rich regions allow a small degree of bending in the DNA, which strengthens protein-DNA interactions. EcCRP does not directly “read” the base sequence at positions 1-3 and 20-22, instead, contacts between the protein and the phosphate backbone of the DNA are favoured when these positions are A/T rich because A/T runs are more flexible. Thus, there has been no reason to think that it matters whether there is a T or an A at position #1 (for example); either base will do. However, Heather made the great point in lab meeting that covariance between adjacent bases at positions 1-3 and 20-22 may be important and would not be apparent in a sequence logo. In other words, if position #1 is a T, it may be much better for CRP binding if position #2 is also a T; conversely, if #1 is an A, position #2 is more likely to be A.

I used MatrixPlot (which has been previously used in our lab for USS analysis, see here) to detect covariance in the 45 experimentally determined H. influenzae CRP sites. Absolutely no covariance was detected between any positions over the 22bp length. I think 45 sites should be adequate for detecting significant covariance. This result is good news because it means that when I make a point mutation in ICAP, I probably don’t have to worry that this same mutation would have a dramatically different effect on CRP affinity if it were introduced into a different site (as I had speculated before). In other words, base contributions to CRP binding may be additive and not cumulative, which makes experiments more straightforward and easier to interpret.


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