Question: 7. The Single Stranded Binding protein (SSB) is a monomeric protein with a molecular weight of 35,000 g/mol. It binds tightly to single-stranded, but not double-stranded DNA. Binding saturates at a 1:12 weight ratio of DNA to protein.
a. At saturation, what is the ratio of nucleotides of single-stranded DNA to molecules of SSB protein? (The average molecular weight of a single nucleotide is 330 g/mol). Express this as “x:1” where x is the number of nucleotides per molecule of SSB.
b. When the binding of SSB protein to DNA reaches saturation, are adjacent monomers of SSB protein likely to be in contact? Assume that a monomer of SSB protein extends for 12 nm along the DNA upon binding and that the spacing of bases in single-stranded DNA is 10 nucleotides per 3.4 nm. Given your answer to (a), how tightly packed are SSB molecules on single stranded DNA when saturated?
The binding of SSB protein to DNA can be analyzed by centrifugation through sucrose gradients, on which the much more massive DNA sediments more rapidly than protein and is consequently found closer to the bottom of the gradient (see page 165 in your book). When analyzed in this way, the binding of SSB protein to DNA shows a peculiar property that is illustrated below. In the presence of excess single-stranded DNA (10 µg), virtually no binding is detectable at 0.5 µg SSB protein (part A of the figure), whereas almost all of the SSB is found bound to 10 µg of single-stranded DNA at 7.0 µg SSB protein (part B of the figure).
c. Given your calculations, and the experimental data shown above, can you describe how you think SSB molecules interact with DNA and with each other?
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