Current LectureMCB 250 1st Edition Lecture 4Outline of Current Lecture 1. Forces for DNA structure2. Major and Minor groove3. DNA is dynamic4. Differences between structures5. Chemical and Physical properties of DNA6. Noncovalent bondsCurrent Lecture1. All forces are responsible for DNA structure- Base Pairing: H- bonds. Strength of H bonds is dependent on its position. G C has 3 H bonds, A T has 3 H bonds. Fit to maximize H bonds to find relative position.- Base Stacking: Aromatic bases are flat wanting it to interact in a parallel fashion so electron clouds can interact with one another. - Once H bonds are fulfilled within the base pairs, we have aromatic rings and H bonds arefilled and they don’t need to interact with water anymore. Then the base pairs themselves are hydrophobic. The hydrophobic regions of bp want to stay together (inside)o Phosphate and sugar are hydrophilic because it is charged on the outside- Phosphates are negatively charged. Each base has one phosphate (phosphodiester bond)link to sugar, each strand has negative charge in each side; the negative charges are repelling one another. This drives the phosphates to the outside. o Negative charges can’t exist by themselves. They are counteracted by cations (Mg2+) (divalent 2+) to interact with the structures of DNA. This is never drawn in though. The salts are there though.- The combination of all these forces that gives rise to the structure.- Ribose 5’ C is up top hooked to phosphate and 3’ hydroxyl group. This allows the difference of 5’ and 3’.2. Major and Minor GrooveThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- For every base pair, there’s a major and minor grove associated with it each bp on each side. This is because the bases are attached to the sugar in an angle. Every base plugs into major and minor groove.- Accessibility of major groove is more extensive than the minor groove- There’s more information on major groove than minor groove. There’s information for specific proteins that can read and recognize the DNA specific sequence.o It reads the H bonding groups that extend in primarily the major groove- GC base pair: AADH in major looks different in CG base pair HDAAo The minor groove is ADA both ways, you cannot distinguish between CG and GC bp in the minor groove, but in the major groove- AT: ADAM (methyl group)o Minor groove is AHA.- You can distinguish between AT and GC base pair in minor but not that much information.3. DNA is dynamic:- Things are always moving around. o Base stacking: base pair on top and base pair on bottom for bp in middle. Chances the middle one breaks off is rare. It is being held in place in each side. The end, last bp is missing it in top.- Doesn’t stay still. The single bonds rotate. The base pairs at the bottom are hydrogen bonded and shakes around and breaks off. Because it is not stacked it comes off but gets back to it. - Some DNA are straight, some are naturally bent. There is a sequence of runs of AAA separated by 10 bp (one turn) then the same face if back and it bends the DNA. Some DNA are naturally bent. - Proteins can also bend DNA. Histone: Proteins that DNA is wrapped around. The DNA is bent around the histone molecules. They are non sequence specific that bond to the phosphates.- Propeller twist: Sequence of DNA can have the bases twisted a little to have a propeller. Somewhat sequence specific- Non Sequences Dependent structures B form DNA in other structures- Cruciform DNA: Sequence palindrome. Same sequence 5’ to 3’. The piece of DNA can unfold and you can have base pair interaction within the same strand to form a cruciform from a straight line.o Doesn’t happen all the time, depends on chemical stability- 4 G’s can come together (G quadruplex) (more complicated)o Metal ion in the middle (mg+)o A lot of G’s in a certain length, it can fold up into a G quadruplexo If it is stable enough, it can drive the reaction to the regular DNA.o Proteins can recognize the sequence to carry out a function4. Differences between structures:- Can occur due with different sequences under different conditions- A form DNA: RNA o Can change conditions in test tube to drive DNA into the structures of A and Z DNA.5. Chemical and physical properties of DNA- Bases of the DNA are aromatic compounds and absorb light in the UV range. They arrange with peak of 260 nm.- Changing the environment of the bases will change its ability to absorb light- Environment is different in double and single strand DNA- Use a spectrophotometer- Use light that’s filtered to a single wavelength and shine through a sample and see how much light is coming through the other side - You can monitor absorption of any molecule (DNA) over a series of wavelengths of light -> Spectrographo Different wavelength of light, molecule absorb that light different energies differently. The peak absorption dna is 260 nm. Enough energy to kick electrons from aromatic bases to a quantized state. This depends on the environment. The environment of the bp in DNA is different with ssDNA. Ss bases can absorb more light than double stranded DNAo Increase in absorption from the same amount of DNA from going to double to single DNA. This is the hyperchromic effect (more light/absorption)- In a given molecule has a certain spectrum. Peak absorbance is 260 nm. If you choose 280 nm, 260/280 is always the same for pure DNA.o Beer’s law; the absorption of light in a wavelength is dependent on the concentration and length. o If a piece of DNA at 260 nm is doubled, you’ll absorb twice as much length. If youchange the path length of the cubette (shining light through a material, if the tube is 2x big with same concentration, you’d absorb twice as much light)o It can absorb so much energy in a wavelength. You get ratio of extinction = 1.8. Any piece of DNA ratio at 260 and 280 is always ratio of extinction coefficient 1.8- Use this to quantitate how much DNA there is. You look at absorption at 260nm. Because it is a constant. Measure the E and L and you can concentrate concentration of DNA. o DsDNA will absorb 260 with a unit of 1o Make it to single strand, absorption will go up to 1.37o Free bases can absorb light better of 1.60- By measuring the A260 for dsDNA you can determine concentration.o IF the DNA is pure, it is reliable.o To check this, if its not contaminated with