[RASMB] Protein binding to polymer ( poly(A)-RNA )

[Cole, James]

Dear Christoph-
For nonspecific protein-nucleic acid binding, the stoichiometries as a function of RNA length should increase with a "staircase" pattern. They will increase by one when the RNA length equals a integral multiple of the protein binding site size.  If you want to interpret the apparent binding constants, you need to take into account the fact that that the protein is "large" in that it covers more than one base, giving rise to apparent anti-cooperative binding.  Luckily, there is well established theory for analyzing these systems. The original model wAS developed by McGhee and Von Hippel in the 1970s for infinite lattices (1). This model has been adapted for finite lattices and  competitive binding (2).   There is a also a simple combinatorial model for binding to finite lattices (3).   We adapted Epstein's model to cover cases where the protein can overlap along the nucleic acid lattice (4).  Michael Fried has also looked at a system where the proteins overlap along a dsDNA lattice (5) producing this bizarre oscillation in the apparent binding site size and cooperativity parameter as a function of DNA length.

Hope this helps.

Jim Cole

(1) McGhee, J. D. & von Hippel, P. H. (1974). Theoretical aspects of DNA-protein interactions: co-operative and non- co-operative binding of large ligands to a one-dimensional homogeneous lattice. J. Mol. Biol. 86, 469-489.)
(2)  Tsodikov, O. V., Holbrook, J. A., Shkel, I. A. & Record, M. T., Jr. (2001). Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer. Biophys. J. 81, 1960-1969.
(3  Epstein, I. R. (1978). Cooperative and non-cooperative binding of large ligands to a finite one-dimensional lattice: a model for ligand-oligonucleotide interactions. Biophys. Chem. 8, 327-339.
(4) Ucci, J. W. & Cole, J. L. (2004). Global analysis of non-specific protein-nucleic interactions by sedimentation equilibrium. Biophys Chem 108, 127-140.
(5)  Melikishvili, M., Rasimas, J. J., Pegg, A. E. & Fried, M. G. (2008). Interactions of human O(6)-alkylguanine-DNA alkyltransferase (AGT) with short double-stranded DNAs. Biochemistry 47, 13754-13763.




On 4/6/10 6:40 AM, "Christoph Brockmann" <chbrock at zedat.fu-berlin.de> wrote:

Hi Everybody,

I got myself into an interesting problem in that I am trying to
characterize the binding of a small protein domain to poly(A)-RNA. I am
getting fairly descent data for defined length RNA oligos using ITC, but
have serious problems fitting this data to the usual models. It fits,
but the stoichiometries (although clearly dependend on the polymer
length) don't make a lot of sense to me.

Is there an established theory that can be used to describe binding
events with multiple similar binding modes? Since we don't know the
exact optimal length of the RNA, we are almost certainly using RNAs that
are either too long or too short, giving rise to multiple register
binding either way.

Any help would be very much appreciated.

many thanks,
Christoph
_______________________________________________
RASMB mailing list
RASMB at rasmb.bbri.org
http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb


----------------------------------------
James Cole
Associate Professor, Department of Molecular and Cell Biology
Head, National Analytical Ultracentrifugation Facility
University of Connecticut
91 North Eagleville Road
Storrs, Connecticut 06269
Tel: (860) 486-4333
Fax: (860) 486-4331
email: james.cole at uconn.edu