Life Science 3 Lecture 3

Front 1

How is genetic information contained in the DNA processed into usable information?

Back 1

genes--> proteins
sequence of nucleotides-->sequence of amino acids

The first experiment to shed light on this process used a technique commonly referred to as a Pulse-Chase experiment. All about Pulse and then Chase.

Genes are segments of the DNA are defined by sequenc of nucleotides. DNA is made up of building blocks of nucleotides.

Front 2

Pulse-Chase Experiments

Back 2

Pulse: short exposure to labeled precursors

Synthesis of DNA, RNA or ptoein involves the polymerizaton of precursor molecules (nucleotides or amino acids)

If these precursors are radioactively labeled and supplied to cells in a culture media, then the cells will transport them into the cell and use them to synthesize the macromolecule.

If cells are examined immediately for the location of the radioactively, the site of synthesis can be determined

Nucleotides are precursors to DNA: if you want to incorporate label into DNA-if you gets incorporated into long molecule.

If radioactively labeled and supplied to cells then cells will transport them into the cell and use them to synthesize macromolecules.

Hershey Chase: feed phosphate or sulfate and this was getting incorporated into building blocks then macromolecules.

Look at this incorporation immediately or let it go into a chases and see where they move

Front 3

Pulse-Chase Experiment

Back 3

Position of macromolecules in cell are fixed and free: amino acids are washed away. Autoradiogram detects location of labeled protein.

Cell and nucleus: now have radioactive amino acid: gets taken up by the cell and when cell makes proteins-it incorporates these radioactive amino acids into length of protein

If we want to visualize this we don't want alot of unincorporated amino acids floating around

fix cells on a microscope slide and use some fixitive that will cross link it across slide: small molecules can rinse right off. Left only with radioactivity where its incorproated into large macromolecule

Front 4

Pulse Chase Proteins

Back 4

X ray film is dark where radioactive emissions are detected. In this experiment, protein synthesis is detected in the cytoplasm but not the nucleus

This is what happens when you use S-36 methionine (amino acid) very specific for proteins: center spot is in the nucleus and the rest is the cytoplasm. Can see after 15 minutes exposure to radioactive materials they can find all kind of proteins being made in cytoplasm but nothing in the nucleus

Conclusion: In eukaryotic cell proteins are made in cytoplasm

Front 5

The Chase Experiment

Back 5

-Wash or dilute out label and allow the cell to continue growing for a period
-During this period no new incorporation of radioactive precursor in macromolecule occurs; however, macromolecuels may move above the cell
(determines final location of macromolecule)

Chase experiment: was hor dilute out label and allow cell to continue growing for a period
During this period no new incorporation of radioactive precursor in macromolecule occurs; hwoever, macromolecule may move about the cell [where did it go? Stay int he same place or move about the cell]

Front 6

3 Pulse Chase Experiments

Back 6

-Grow cells in presence of labeled precursor (this allows the labeled compound to become incorporated into the macromolecule during synthesis)
-Autoradiogram of pulsed cells
-Wash remaining cells, grow in presence of unlabeled precursor
-Autoradiogram of chased cells

35 S-methionine: protein
3H-uracil: RNA
3H-thymine: DNA

Shows where everything is being synthesized

Front 7

Results of the Pulse Chase Experiments

Back 7

-35S- Methionine: Protein in cytoplasm, chase goes into nucleus
3H-Thymine: after pulse is in nucleus, amde in nucleus and stays in nucleus

Problem: DNA directs synthesis but they are not in the same compartments: DNA in nucleus and protein synthesis in cytoplasm

Uracil: RNA made in nucleus but give it time moves into cytoplasm

Led to hypothesis that RNA is made using DNA as its template and then DNA is converted to RNA and RNA takes genetic informatoin and moves it to the cytoplasm where it can be used to synthesize proteins.

This is where RNA got name messenger RNA

Front 8

Conclusions about Pulse Chase Experiments

Back 8

-Protein is synthesized in the cytoplasm, and later may move to the nucleus
-DNA is synthesized in the nucleus and remains there
-RNA is synthesized in the nucleus and migrates to the cytoplasm

Hypothesis: RNA carries genetic information from the DNA to the site of protein synthesis

Front 9

Central Dogma of Molecular Biology

Back 9

RNA carries genetic information from the DNA to the site of protein synthesis

DNA transcribed to RNA and then RNA is translated into protein. This process is known as a gene expression. It is unidirectional.

There are instances where RNA can go bacK to DNA

Front 10

Environmental Signals and Central Dogma of Molecular Biology

Back 10

Having this complex process allows for a lot of areas where you can regulate: alot of different signals can affect transcription; stability; rate of translation and influence protein after it is synthesized

A whole lot of places where we can regulate gene expressions

All cells have exactly DNA: different tissue types express different proteins: DNA isn't different in cells but its the expression of DNA into proteins

Front 11

RNA vs. DNA (4)

Back 11

-Ribose instead of deoxyribose in the backbone [less stable in presence of alkali] (differ in 2; position of oH
-Uracil instead thymine [5-methyl-uracil
-Single stranded
-secondary structure: bound by proteins to keep it stretched out

same 5' and 3' ends; same phosphodiester bonds

Front 12

why is RNA less stable in alkali then DNA

Back 12

When you subject RNA to base; you will ionize this oH and this O becomes a nucleophile that will attack phosphate; breaks the phospho-oxygen bond and end with cyclical molecule and the backbone is broken

You can use knowledge to separate DNA and RNA; add base and the RNA will be automatically degraded into nucleotide.
RNA is less stable then DNA into physiological information; may be why DNA is now genetic information

Front 13

Uracil vs Thymine

Back 13

-Thymine and Uracil are very similar
-Only difference is a methyl group (in thymine) instead of a hydrogen group
-Doesnt change base pairing properties
-Helps enzyme recognize what should be in DNA or in RNA

Cytosine has an amino group: if it gets deaminated it turns into uracil. Why thymine is in DNA instead of uracil

Front 14

RNA structure

Back 14

-RNA is primarily single stranded with regions of secondary structure
-Double stranded regions are in A, not B form
-All bases have hydrogen bonding potential
-If you have regions complimentary then will bond to each other
-At top bases won't be bonded to anything but water but at bottom form of helix [hairpin]

Can also get two sections: double helical with single bulge [bulge] or double bulge [loop]

Base pairing can also occur between non-contiguous regions [pseudoknots]

RNA that carry message are usually coded with proteins and ribosomes so can't fold into complicated structure but others are always found in 3D structure

Front 15

Uracil can also pair with...

Back 15

guanine: slipped it up and allow these donors and acceptors to bond rather then slide down instead of bumping into each other

Front 16

Is triple base pairing observed in RNA?

Back 16

yes-can help stabilize many 3D structures

Even have three different bases: hard to predict
not many rules about where it is 3D
potential for all kinds of structures

Front 17

Double helical character

Back 17

Fold-back regions have double helical character: regions that do fall back can start twisting around into a helix

Front 18

Purpose of 2 OH

Back 18

2' OH in ribose causes RNA to favor the C-3' endo because this facilitates base pairing
2' stabilizes; hydrogen bond to oxygen in ring of the next nucleotide
This is because oH and there for hydrogen bonding

Front 19

Role of ions in secondary structure

Back 19

Mg2+ and K+ stabilizes secondary structure
alot of phosphates or molecules

Front 20

Can RNA catalyze biochemical reactions?

Back 20

RN that can cataylze a reaction, cleave itself
Very thick structure, folds itself into structure and does a cleavage reaction
THe discovery: theory that the world could ahve evolved from an RNA

Front 21

Evidence that life evolved from RNA world

Back 21

Everything in the living cell can be performed by RNA:
1) contain genetic info and have a code
2) fold into 3D structures and catalyzes reaction
3) replicate itself

The simplest world could have been an RNA world: proteins evolved because could make more structures more stable

Front 22

Classes of RNA first discovered

Back 22

1) RNA associated with ribosomes: ribosomal RNA or rRNA
2) small abundant transfer RNA or tRNA
3) variable, less abundant: messenger RNA or mRNA [found when studying bacteriophage]

T4 Experiment: In cells after they are infected by T4 pahge they discover that synthesis of all this other RNA's stopped and new class of RNA was synthesized. Complimentary to the T4 genome. Become associated with ribosomes and tRNA to direct protein synthesis
Study of T4 was discovery of messenger RNA
Easier system because very small genome and produces alot of RNA in response to infection

Front 23

Protein structure

Back 23

-Proteins are polymers made up of amino acids
-There are 20 different amino acids: each with different chemical properties
-The 3D shape a protein adopts is determined by its sequence of amino acids
-The large number of 3D shapes that proteins can assume allows them to perform a wide range of functions

Front 24

Amino acid general section

Back 24

zwitterionic: + and - charge simultaneous

Front 25

Polar negatively charged R groups

Back 25

Aspartate (Asp-D)
glutamate (Glu-E)

amino acids are in aqueous enviornemtn: lost proton and are negative charged; have additional negative charge

both carboxyl, ionized, negatively charged

Front 26

Basic amino acids (positive)

Back 26

-pick up proton under aqueous environment and have a positive charge

Histidine (His-H)
Lysine (Lys-K)
Arginine (Arg-R)

conjugate acids (+)

Front 27

Nonpolar, aromatic R groups

Back 27

Phenylalanine (Phe-F)
Tyrosine (Tyr-Y)
Tryptophan (Trp-W)

hydrophobic
absorb light

Front 28

Side chains typically involved in H bonding

Back 28

Serine: (Ser=S)
Threonine (Thr=T)
Cysteine (Cys=C)
Asparagine (Asn=N)
Glutamine (Gln=Q)

polar uncharged: have ability to form hydrogen bonds
also tyrosine

Front 29

Hydrophobic amino acids

Back 29

These side chians do not like to be in aqueous environment

Glycine (Gly-G)
Alanine (Ala-A)
Proline (Pro-P)
Valine (Val-V)
Leucine (Leu-L)
Isoleucine (Ile-I)
Methionine (Met-M)

all have methyl groups/H

Front 30

Peptide bond

Back 30

formed by joining the amino end of one amino acid to the carboxyl end of another amino acid [lose water]

peptide bond

form backbone of proteins giving it certain characteristics: can restrict how a protein can fold

Front 31

polypeptides

Back 31

monomer: polymer
polypeptides are linear polymers of amino acids; peptides usually contain less than about 20-30 amino acids
proteins usually contain 100-2000 amino acids

Front 32

Resonance of peptide bond

Back 32

Gives partial double bond character: gives planarity and resonance

Front 33

Zwitterionic

Back 33

Backbone is uncahrged except for the N terminus (NH3+) and C terminus (CO2-)
NH and CO in backbone are available for H bonding

any charge it will have to come from side group (acidic or basic will provide charges)

Front 34

Bonds in proteins

Back 34

-Covalent bond hold the amino acids together, non covalent bonds are responsible for the 3D structure

hydrogen bonds
ionic bonds

van der waals interaction and hydrophobic bonds: ionic bonds can only form at ends or charged R groups or can interact with water

van der Waals and hydrophobic are driving force: for how a protein is going to fold
getting groups out of water provides energy

If R group is hydrophobic wont hang out in aqueous but instead will want to fold in and form hydrophobic core: most have hydrophobic anmino acids on center that is hidden away form awter

huge force is hydrophobic side chains trying to get away from center; away from aqueous environment