深挖坑 广积水 不刷屏

VacuumUID: 2200326

真是太可怕了！第一次见这么灌的！！坑好深啊！！！

VacuumUID: 2200326

真是太可怕了！第一次见这么灌的！！坑好深啊！！！

VacuumUID: 2200326

真是太可怕了！第一次见这么灌的！！坑好深啊！！！

VacuumUID: 2200326

真是太可怕了！第一次见这么灌的！！坑好深啊！！！

VacuumUID: 2200326

好奇怪！都灌了那么多水了，还是新手上路，嘿嘿！快成为新兵吧！！！

VacuumUID: 2200326

突然发现已经是新兵了！好开心啊！

VacuumUID: 2200326

都是自己的名字，好开心啊
嘿嘿

premrnaUID: 2124906

For further reading on effects of sequence on structure,

“Understanding DNA-The Molecule and How it Works”
By Calladine and Drew


Major Conclusion:  DNA structure can depend on sequence
In predictable, yet complicated ways.

Therefore, DNA binding proteins can recognize structure,
And they can be designed to bind to highly flexible DNA.

premrnaUID: 2124906

1.  If you were to mix the bases of DNA with water, would you expect them to form co-planar hydrogen bonds, or stack on top of each other.  What about in an organic solvent such as DMSO?  (Hint, the answer is different in each case).  Justify your answer.

premrnaUID: 2124906

In water, they would stack, because there is a major entropic advantage to removing the hydrophobic interactions from the water .  In DMSO, stacking is not favored because there’s no entropic cost to not doing it.  In addition, if you make co-planar base pairs, you are able to undergo hydrogen bonding.

premrnaUID: 2124906

2. This question addresses the effects of salt on DNA-binding by  repressor.  When  repressor binds to DNA, it displaces 5 ~ ions from the phosphodiester backbone of DNA (Sauer and Pabo, Adv. Prot. Chem 40, 1-61).

premrnaUID: 2124906

A.  What is the Gion release in 50 mM KCl?   

Use Sion release = R ln [Cb/Cf]        Cb = [ions] on DNA (~2 M)        Cf = [ions] in solution
To get the free energy:   Gion release = - T Sion release   
Assuming Cf = 50 mM, each mole of displaced ions gives ~ -2.2 kCal/mol
To get total free energy change, multiply by the number of ions released.
Answer:  -11 kCal / mol  

B.  What happens to  Gion release as we increase the salt concentration from 50 mM to 300 mM?  Also express your answer in terms of the effects on the equilibrium constant.

Gion release(50mM) = -11 kCal / mol         Gion release(300mM) = -5.6 kCal / mol
This translates into a change in Kdiss of 3 orders of magnitude!!!
Protein:DNA interactions are exquisitely sensitive to salt concentrations.

C.  At 50 mM KCl, GTotal (the total free energy for lambda  repressor binding to DNA) is -17 kCal / mol.  Given this information, what is the maximum “specificity ratio” between operator DNA and random DNA that can be achieved for lambda repressor?

Gion release = -11 kCal / mol                   But,  GTotal = -17 kCal / mol!!

Therefore,  Kdiss = 3.5 x 10-13   But:  Kion release= 9 x 10-9 arises from interaction with any DNA sequ.

The ratio Kion release/non-specific / Kdiss/specific = 26,000

This shows that there is a fundamental limit on how selective a DNA binding protein can be.

premrnaUID: 2124906

3.  You do PCR with two primers, one of which contains a mutation.  Assuming 100% efficient PCR (all molecules act as templates in every cycle), what % of strands contain the mutation after 3 cycles?  What about after 7 cycles?   Justify your answers .

premrnaUID: 2124906

Since amplification of the wt strand is linear (one new strand made per cycle), there are 2+3 wt strands after 3 cycles and 2+7 wt strands after 7 cycles.  The total number of strands is 24 = 16 after 3 cycles and 28 = 256 after 7 cycles.  3/16 = 18.75%  and 9/256 = 3.5%.

premrnaUID: 2124906

4.  Explain the difference between  plectonemic and solenoidal supercoiling.