Lec 3: More DNA, RNA, Boltzmann Factor (and

Proteins?).

Homework

HW#1: Due MondayReading, ECB, Chpt 1: Due Wednesday.

(Don’t worry about microscopy details. We’ll cover this later.)

Quiz next Wednesday

• Difference between 3’ and 5’ ends? (definition of directionality of sugar)

• Why is bond strength different between base pairs? (# of H-bonds)

• Gene-can have number of nucleotide not divisible by 3? (No)

• Know more about-how a protein finds a binding site? (Have special lecture)

• Why do RNA & DNA not share all 4 bases? (T is better than U for long-term storage!)

• Different types of RNA...”small RNA”?• Energy Landscape of protein folding…

(More later lecture.)• A-T = 2kT; G=C 4kT. Difference of one H

bond but leads to 2kT. (Energy can vary depending on what’s around it.)

Questions from last timeMany excellent questions.

They are explained throughout today’s lecture

-

If add salt to solution, what is effect on melting Temp?Melting temp = Temp. at which DNA strands come apart.

3’

5’

[salt]↑; Charge repulsion ↓ ; Tm ↑

Difference between 3’ and 5’ ends?

– stacking keeps it together (Grease);Phosphate negative charge makes it water soluble (Soap)

Boltzman factor + Partition function(review of basic Stat. Mech. – see Kittel, Thermal

Physics

iE / kTiP E const. e

E1

E0

Temp, T

If T = 0 ºK, what proportion of particles will be in E1, Eo?

If T > 0 ºK, what proportion of particles will be in E1, Eo?

Answer: pop(Eo) = 1 pop(E1) = 0

iP E 1

j

N-E / kT

j=0

1const.= 1/ Z

e

Z = partition function j

N-E / kT

j=0

e

j = represents jth state

iE |kTi

1P E e

z

e-(E1-Eo)/k

BT

Partition Function for 2-state system

1

o 1

1

o

-E /kT

1 -E /kT -E /kT

- 1 E

- o E

eP E =

e +e

o

1

E = mgh

E h = 0

E h =(mg)(h meter)

P(h) =

P(0)

Simple case: Ball in gravitational field.

Thermal fluctuations, finite probability of being at height, h.

E = ??

As ball gets smaller, probability at height h gets smaller / larger ?

“Ball” the size of O2? Why can you breathe standing up?

For O2, 1/e height is ~10 km ~height of Mt. Everest.(10 km is “death zone”)

Probability of dying if you go over 20,000 ft is 10% for every trip!!

What is 1/e height for O2?

-mgh/kT e

kT=4 pN-nm

Two states A – B bonded: E ~ -5 kT (a few H-bonds)

A , B not bonded: E = ??

A + B A – B

5 /5

/,

148kT kT

A Bo kT

A B

P ee

P e

149 molecules148 will be A - B1 will be A, B

0

If you have many weak bonds (e.g. each bond only few kT) you can get a biomolecule that will not fall apart.

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Zipped vs. unzipped

-2 1e ~ 8What if 10 weak bonds?

H bonded ~ 2 kT

What if just one bond? Bond/unbound?

DNA double helix: Many weak (H-bonds), makes for very stable structure.

Many base pairs, essentially completely stable. Still have end-fraying, but probability that whole thing comes

apart– essentially zero. [Need enzymes to separate.]

-20e

With proteins, lots of hydrogen and weak bonds – have conformational dynamics, but rarely fall apart!

Need to know Chemical Bonding

– 100kBT. Sharing of electrons. C-H

–kBT (weakest, but many of them together--significant). Two neutral atoms have instantaneous dipoles, and attract.

1. Hydrogen attached to a very electro-negative elements, (O, N) causing the hydrogen to acquire a significant amount of positive charge.

2. Lone pair– electrons in relatively small space, very negative.

Result is H is (+) and O is (-). Will bind to other molecules

www.chemguide.co.uk/atoms/bonding/hbond.html#top

Neon: -246°C; Xenon: -108°C

Is light enough to break covalent bond?1m=1eV; kBT = 1/20eV. 1m = 20kBT (close)

– varies tremendously, 100kBT to few kT. + and – attract, but depends on solvent.

Na+ Cl- = few kBT (break up easily)– few kBT, up to 5kBT

4 types: what are they?1. Covalent

2. Ionic

3. Hydrogen

4. Van der Waals

Size Scales of DNA (+ Protein)Chromatin = Complex of DNA + Protein (histones + non-histones)

3 x 109 = 3 billion

# chromosomes?

Length/chromosomes?

~ 1/50 meter = 2 cm!

46 (ca. 50)

8/17/06

~ 1 meter

Flexibility of DNA?

~ 1 meter packed in3-10 m (size of nucleus)

Nucleotides

[4 Diff. types, A,T,C,G]

Bacteria - 1 m Eukaryotic cell – 10-100 m

1 m10-30 m 10-100 m

(Nucleus 3-10 m)

Cell Size

Interesting factoids:1. 1014 cells in body…

…more stars than in Milky Way Galaxy.

2. 200 different types of cells in body.

So a meter of DNA must pack 3-10 m!How much DNA inside of every single cell? 1 meter

What does this tell about bendability of DNA?Like spaghetti, uncooked or cooked?

See how this is measured using magnetic tweezers

DNA RNA ProteinsCentral Dogma of Biology

http://learn.genetics.utah.edu/units/basics/transcribe/

DNA: series of 4 nucleotides (bases): A,T,G,C

RNA: series of 4 nucleotides (bases): A,U,G,C

Transcription [DNA & RNA similar]

Translation [RNA & Proteins different]

Proteins: series of 20 amino acids: Met-Ala-Val-… each coded by 3 bases amino acid

AUG Methionine; GCU Alanine; GUU ValineProteins are 3-D strings of linear amino acids

Do everything: structure, enzymes…

Question from last time

Answer: Yes, small molecules move through diffusion. However, mRNA is most likely bound by many proteins forming an RNP complex and this is large enough to prevent free diffusion. The nuclear pore complex has mechanisms to control the influx and outflux and this is still being worked out. Have you heard of RanGTP etc?

Q: How does mRNA (and tRNA) leave the nucleus after it is synthesized? Does it move through the pores simply by diffusion, or is there some active transport machinery?

Q: How, when it comes to making RNA, how does it “know” to use U instead of T? I gather that the extra size of T--it has an extra CH--is enough to cause T not to bind to the RNA polymerase, whereas U can?

Answer: RNA polymerase probably cares more about the presence or absence of the hydroxyl group at the 2' position to distinguish between U and T, but this is just my guess.

Ran (RAs-related Nuclear protein) is a small 25Kda protein that is involved in transport into and out of the cell nucleus during interphase and also involved in mitosis.

Ran is a GTP binding protein that is essential for the translocation of RNA and proteins through the nuclear pore complex. Because of its many functions, it is likely that Ran interacts with several other proteins.http://en.wikipedia.org/wiki/Ran_%28biology%29

Difference between RNA and DNA is the Sugar + 1 Base

RNA is a string of nucleotides, just like DNA

RNA substitutes Uracil for Thymidine

Uracil will base pair with many groups.Methyl group restricts uracil (thymine) to pairing only with adenine.

This greatly improves the efficiency of DNA replication, by reducing the rate of mismatches, and thus mutations.

Also, methylation protects DNA from viruses.

Larger groove (than DNA)—more likely to be attacked by enzymes)

Difference between RNA & DNAFrom Nobel Lecture

The discovery of catalytic properties in RNA also gives us a new insight into the way in which biological processes once began on this earth, billions of years ago. Researchers have wondered which were the first biological molecules. How could life begin if the DNA molecules of the genetic code can only be reproduced and deciphered with the aid of protein enzymes, and proteins can only be produced by means of genetic information from DNA? Which came first, the chicken or the egg? [Sid] Altman and [Tom] Cech have now found the missing link. Probably it was the RNA molecule that came first. This molecule has the properties needed by an original biomolecule, because it is capable of being both genetic code and enzyme at one and the same time.

Presentation Speech by Professor Bertil Andersson of the Royal Swedish Academy of Sciences, December 10, 1989

RNA can be catalytic!1989 Nobel Prize—Altman & Cech

Class evaluation

1. What was the most interesting thing you learned in class today?

2. What are you confused about?

3. Related to today’s subject, what would you like to know more about?

4. Any helpful comments.

Answer, and turn in at the end of class.