Schleif's Questions, 2018
March 6, 2018
4.(9pts) How many base pairs of DNA would it take to tie up an E. coli like a Christmas package (bow not required)?
Length of a cell is a micron or so. Therefore total length required is arount 3.4 microns. At 3.4 Angstrom/bp, this is 3.4 x 104/3.4 = 10,000 base pairs.
5.(9pts) When a single RNA polymerase initiates transcription and proceeds down the DNA, positive supercoiling is generated in front of the polymerase. With respect to supercoiling what happens when two, then three, and finally a whole series of RNA polymerases initiate one right after the other?
A transcribing RNAP generates positive supercoiling ahead of itself and negative supercoiling behind. The positive supercoiling ahead of each successive polymerase neutralizes the negative supercoiling left behind by the preceeding polymerase. Hence, there is no net effect beyond what is generated by the first RNAP.
6.(8pts) Devise a regulation mechanism in which binding of the activator to DNA stimulates transcription, but where the activator does not touch RNA polymerase or any other protein in the process.
Almost everyone answered that the activator could stimulate DNA looping so as to bring another activator into contact with RNAP. Also a lot answered that the activator could change DNA supercoiling in its vicinity, which is hard for me to understand, but I gave full credit for this anyway as well as full credit for the looping answer.
7.(8pts) Textbooks and Google say that pyrophosphatase is required to make the reactions of RNA and DNA synthesis irreversible, and yet, one can perfectly well synthesize RNA and DNA in vitro in the absence of pyrophosphatase. Who is right, the experimentalists or the writers? Why?
In the confines of a cell, the pyrophosphate concentrate will build up and push back on the dNTP incorporation reaction that releases a phyophosphate. In vitro, reaction conditions are much much more dilute and the pyrophosphate concentrations generally do not become high enough to inhibit the forward reaction.
8.(8pts) In the study of proteins that bind to DNA, when is it possible to use the dissociation rate constant (easily measured) to estimate the equilibrium dissociation constant (much more difficult to measure)?
When it is known that the forward rate constant of binding to DNA is pretty much constant, which it is for most DNA binding proteins, and in fact is almost always near the diffusion limit. In these cases, since Kd=koff/kon, Kd and koff are proportional.
9.(8pts) Bacterial mutants lacking the origin of DNA replication, OriC, obviously cannot grow. Surprisingly, mutants can be found that do grow, albeit very slowly. These are found to be defective in RNAseH. What is going on or what do you think is going on?
Fragments of RNA left from aborted RNAP initiations or breakage of RNA in the process of its synthesis or merely pieces of RNA that displace a strand of DNA then provide 3'OH groups in position to initiate DNA synthesis by the polymerases usually utilized for DNA repair. RNAseH, which digests RNA only in RNA-DNA hybrids prevents this spurious initiation. Hence, in an OriC negative cell also lacking RNAseH, DNA synthesis adequate to support a very slow growth rate can occur.
Schleif's Questions, 2018
April 17, 2018
10. Suppose you have the 3D structure of a human protein that for industrial purposes, you wish to be active at 45 degrees C. The native protein however, is denatured at this temperature. What changes or kinds of changes would you look to make to increase the protein’s thermal stability?
Introduce cysteines in positions compatible with formation of disulfide bonds, increase polarity of surface amino acids, reduce polarity of buried amino acids, neutralize or eliminate any buried charges.
11. Suppose that when purified, an enzyme is fully active, but after storage for a month at 4 degrees, it has lost more than 90% of its original activity. The enzyme does not use any cofactors. Mass spectroscopy of the protein shows that no covalent changes have occurred (like oxidation of a methionine or formation or breakage of a disulfide bond). What do you hypothesize has happened to the protein, and how might you acquire additional evidence to support your hypothesis?
Hypothesize that protein initially folds into a state that is not the lowest energy, but with time, transitions to a lower energy conformation, but one which is not active. Demonstrate by denaturing and refolding, upon which the activity should be restored, only to disappear again on storage.
12. The following drawing was found in the notes of a young genius researcher investigating transcription factors. Unfortunately, the comments following the figure have been lost. Write what you think they were. The closer your comments come to explaining the salient points of the figure, the better your score.
There are two basic mechanisms for modulating DNA binding affinity of a dimeric protein. Either you change the conformation of the DNA binding domains (A), or you change the spatial positioning of the DNA binding domains (B)
13. Suppose virtually any amino acid change in the N-terminal 20 amino acids of AraC results in constitutivity (operon fully expressed even in the absence of inducer arabinose). A. What is the best explanation for this? B. Suppose that analogous mutations are not found anywhere else in the protein. Comment.
A. The arm is responsible for holding the protein in the DNA looping state, say, by interacting the with DNA binding domain. B. If the arm does interact with the DNA binding domain, similar constitutive mutations ought to exist in the DNA binding domain. The absence of such suggests that the simple answer given in A is incorrect.
14. How would you “look” for a peptide, of length, say 40 residues, that in the absence of arabinose is unfolded, but in the presence of arabinose tightly folds around the sugar? (As should be obvious, the better your scheme, the more points you get. Hence saying that you will look in your grandmother’s cookie jar won’t earn you a lot of points.)
This requires a trivial modification of the method so elegantly described in one of the assigned readings. Use hundreds of thousands of plasmid that code for a surface protein fused to random DNA sequence coding for 40 amino acids fused to GFP. Cells expressing the peptides are treated with arabinose and protease (digests only unfolded protein) and use fluorescent activated cell sorting to obtain those coding for a peptide that is folded in the presence of arabinose. These are pooled and arabinose is removed and cells are again treated with protease. This time collect the cells that have lost the GFP.