Lecture 13

Question 1

Central dogma:

Answer 1

DNA->RNA-> protein

Question 2

Does DNA store energy?

Answer 2

No, stores info

Question 3

Polymerization allows synthesis of periodic polymers

Answer 3

Periodic: have a long polymer with many subunits, all the same structure or energy storage forms
Usually individual units of the chain the same, maybe slight differences but not a lot of variety

Question 4

Why are nucleic acids so good for info storage?

Answer 4

Because there’s more variety, nucleic acids are better for info storage

Question 5

Templated polymerization produces aperiodic polymers with defined sequences

Answer 5

Info in the sequence of the polymer itself can lead to its inheritance. Main enzyme to replicate is DNA replicase, DNA polymerase makes a polymer.

Question 6

DNA and RNA are aperiodic polymers that can form double-stranded duplexes

Answer 6

-Hybridization is the pairing of two DNA or RNA strands to form non-covalent duplexes
-formation of hybrid duplexes allows templated polymerization
No info content in backbone- the backbone is identical in each repeating unit
The bases vary from one “repeating” unit to the next- sequence of bases provide the specificity

Question 7

Glycosidic bond

Answer 7

the bond from the N of the base to the C of the ribose (sugar)

Question 8

AMP vs dAMP

Answer 8

The base vs deoxy base

Question 9

phosphoester bond:

Answer 9

O between P and C of sugar

Question 10

anhydride bonds:

Answer 10

O between two Ps

Question 11

A dna duplex

Answer 11

The backbones in a duplex run in opposite directions- they are antiparallel to each other
5’ C attaches to phosphate, 3’ C attaches to hydroxyl group
These two connect and makes linkages in the strand, giving directionality
pKa of the phosphodiester group (3’5’) (in sugar phosphate backbone) is about 1.0. It is always ionized and negatively charged. Later, we will see that counter ions are required to balance these charges.
Key: phosphodiester bond charged because it has low pka, so will always be negatively charged. Negative charges important for structure because they lead to electrostatic interactions. Phosphates can’t get too close b/c they will repel. Need positive charges to balance.

Question 12

How many different kinds of bases in DNA?

Answer 12

Potentially about 8, but some of these are modifications on the original four

Question 13

DNA and RNA hybridization

Answer 13

Hybridization reversible, can denature and anneal again, separate two strands and they will often form random coils. Ss molecule can have parts that are double stranded. Can get intramolecular aggregation (same strand) and intermolecular (diff strand)
key concept is the melting curve

Question 14

Extremely important: Tm

Answer 14

Relative absorbance vs temperature on cooperative curve.
If you raise temperature, ds DNA will convert to ss dna and will increase in absorbance at 260 nm. Tm is the melting point of the DNA. Tm is where half the DNA is ds and half is ss. Steep curve occurs because this is a cooperative process.

Question 15

Factors that control/change Tm:

Answer 15

1) Chain length- longer it is, the more stability. Shifts curve to the right.
2) Solvent conditions (ionic strength, pH). Higher solvent/salt concentrations will increase Tm b/c more ionic charges, strengthens. pH: best is where pH equal to Pka?
3) number of mismatched base pairs- mismatching destabilizes, lowers tm
4) Base composition: GC to AT ratio. High GC content will have a higher melting point and shift the curve to the right.

Question 16

Hybridization: base pairing provides specificity

Answer 16

T and A pair: form two H bonds. G and C pair: form three H bonds. Distance from one phosphate backbone to the other always the same. Important so you get a straight double helix.

Question 17

Watson Crick base pairing

Answer 17

H bonds between Watson-Crick base pairs are important for pairing specificity, but H bonds do not provide the major energetic contribution for duplex formation.

Question 18

Difference between T (dana) and U (RNA)

Answer 18

Extra methyl group on the thymine. Not one of the things differing from base pairing, so if you had U and T in the same place, would not be able to discriminate. C5 methyl does not participate in base pairing.

Question 19

Three dimensional structure of DNA in water- why double helix?

Answer 19

Hydrophobic things have to hide in an aqueous solution. Hydrophobic parts hide from water- double helix puts bases in the middle and hides them.
Ex: Coke- no structure b/c soluble

Question 20

Why is DNA called an acid if it has all these bases?

Answer 20

The phosphates are negatively charged and make it an acid. Overall, molecule is an acid

Question 21

Why DNA prefers to be duplexed?

Answer 21

Hydrophilic sugar phosphate backbone on outside, hydrophobic bases on inside. Can’t just be a straight ladder b/c have a lot of space in the middle. Spaces happen to be about 3 ang long and will allow water to get in. This is a problem. Get a right angle, some rotation around bond but can’t bend at a different angle. Know from bond length that the repeat unit must be 6 and long. Skewed ladder would get rid of space in between (tilt a little). Problem: right angle does not work here, need right angle. The geometry of the glycosidic bond does not accommodate this structure. The base and the sugar are relatively rigid rings. Base stacking can only occur when backbones spiral into a double helix. Rotate 10 degrees, maintain geometry of sugar base bond, can stack these up

Question 22

B form DNA

Answer 22

10 base pairs per helical turn
Watson and crick
excludes water, each stacked up on top of the other
major groove where bases are farther apart, minor is smaller gap
regulatory sequences usually revealed where major groove is, this is where things bind

Question 23

Dimensions of the DNA double helix: B form

Answer 23

Takes 10 base pairs for B form to go one full turn around
Right handed double helix
Know:
Diameter is 20 A
Rise per base pair: 3.4 A
10 base pairs per 360 degree helical turn
and you can derive the helical twist per base pairs: theta = 36 deg
Rise per helical turn: 34 A - 3.4 nm
clockwise = right-handed
found most often in cells

Question 24

A form DNA

Answer 24

When dana is dehydrated. Gets scrunched with a fatter helix. When dna is dehydrated, or if you make ds RNA or RNA/dna hybrids, changes in number of bases per turns and distance (writhe/rise??) changes.
When RNA forms a double helix, it looks more like the DNA a form.in really rich GC content DNA. Question is whether this has any biological relevance- people have identified proteins that only bind to Z form dana so it may

Question 25

Z DNA

Answer 25

Forms a left handed double helix. Big difference. Difference in number of bases per turn only seen

Question 26

The ratio of AT to GC base pairs influences Tm b/c base stacking energies depend on the sequence

Answer 26

What gives DNA stability? not the number of H bonds. Lots of Gs and Cs make DNA more stable. Explanation of hydrogen bonds does not make much sense b/c if you put it in water it will form hydrogen bonds to water. Explanation: force holding DNA together (stacking force). C atoms: VDW distance is about 1.7 A, perfect distance for DNA. VDW are the force between two non charged atoms (induced dipole). Gas and Cs happen to have stronger energy- has to do with how atoms fit together, etc. (beyond scope of the course) VDW are so tiny but there are many so it stabilizes Sometimes has to do with the combination; can have same number of Gs and Cs and get slightly different energies

Question 27

Is the formation of the doubt helix driven by entropy or enthalpy?

Answer 27

2nd law of thermodynamics says entropy of the universe must always increase ss DNA to ds, entropy goes down (less chaos- two strands means a lot more combinations, etc.) Entropy of the universe always increasing, this violates. Overall energy must go up. If you go from ss to ds DNA, you will release heat so you don't violate the second law. Driven by ENTHALPY (heat release), not entropy

Question 28

Polymerization

Answer 28

Polymerization always occurs in the 5 to 3 direction. (SYNTHETIC: USUALLY make an oligo nuc in 3 to 5 dir for labwork, usually in organic solvents)

Question 29

Question: why did nature choose phosphorous?

Answer 29

In ochem, phosphorus is not a good leaving group. Need a catalyst. This is good for biology, where you want to be able to control it (use a catalyst where you want it to leave). Means we can regulate it. Phosphates have goldilocks delta G (50kJ x mol -1) PRPP is first step in purine synth phosphoesters are stable in water (1/2 life= 30 million years), super stable want something that can form linkages to carbon on either side The importance of being ionized Histidine biosynthesis Every single intermediate is phosphorylated until charged amino acid carboxylic acid groups appear. This is a common feature of many metabolic pathways. What about the charge? Keeps it in the cell so it can't diffuse across the membrane. Life first started, it was very important to keep things inside the cell.

Question 30

Rna is not as stable as DNA

Answer 30

B/c of OH group On average, each phosphodiester linkage is hydrolyzed once per 30000000 years. Our diploid genome has 6 billion base pairs per cell RNA backbones break through base catalyzed hydrolysis at least 100 times more often than dna.