Problem Set #9: Protein-Nucleic Acid Interactions

Due November 18 at the beginning of class

Once upon a time, it was hoped that there were simple rules that proteins would use for sequence-specific recognition of nucleic acids; i.e. that one side chain would match a particular base, and thus the protein could “read out” the DNA in a straightforward way. It isn’t that easy, unfortunately.

1. Retrieve the file 1DFM.pdb from the PDB and look at it using a program of your choice. This is the file for an enzyme called a DNA restriction endonuclease. This restriction endonuclease is BglII (nicknamed Bagel-2). It binds DNA at the sequence 5’…AGATCT…3’ and cuts both strands of the DNA backbone between the A and the G, breaking the DNA into two pieces with staggered ends.

(a) What symmetry is present in this structure? Is the protein a heterodimer or a homodimer? Is there symmetry in the DNA? It is observed that restriction enzymes generally recognize palindromic sites, that is, sites that read the same forwards and backwards (like “Madam, I’m Adam” or “A Man, A Plan, A Canal: Panama”). What does that tell you about restriction enzymes in general?

(b) Look at the interface between the DNA and the protein. From the DNA end of things, are most of the contacts in the major or minor groove, or the backbone? From the protein end of things, are most of the contacts from helices, loops, or strands? Does this protein use a recognizable motif?

(c) Draw a map of the contacts between the DNA and the protein. Which are sequence-specific, and which are not? What role to water molecules play in this binding event?

(d) The catalysis in this case is the cleavage of a phosphodiester bond, to generate DNA with 5’ phosphate and 3’ hydroxyl ends. What “metal” ion is involved in this catalytic mechanism? (What other “metals” could be substituted in to serve a similar purpose?) Draw a scheme for the catalytic mechanism.

(e) What are this protein’s closest structural relatives?

2. One of the most important protein-nucleic acid complexes is the ribosome, a huge macromolecular assembly of proteins and RNA that catalyzes the formation of peptide bonds. In all known organisms, ribosomes are where messenger RNA’s (mRNA) and transfer RNA’s carrying amino acids come together in order that protein synthesis will occur. What is especially unusual about the catalytic mechanism, compared to the other proteins we have discussed? How many protein chains are involved in each subunit, and what do they do? (Try the PDB molecule of the month page: http://www.rcsb.org/pdb/molecules/pdb10_1.html).

3. Histone proteins bind to DNA to form nucleosomes using many arginine and lysine side chains. What kinds of interactions do these proteins have with DNA, and why does this make sense considering their function?

4. The measured binding affinity (K_d) of most DNA-binding proteins for DNA is quite sensitive to the ionic strength and pH of the medium. What would you expect to happen to a DNA-protein complex at high salt concentrations and why?