Basics of Molecular Biology Osama S. S. Hassan, PhD MSA Faculty of Biotechnology

Lecture 1 Molecules of life

A- Structure of Water It can be quite correctly argued that life exists on Earth because of the abundant liquid water. Other planets have water, but they either have it as a gas (Venus) or ice (Mars).

The chemical nature of water is thus one we must examine as it permeates living systems: water is a universal solvent, and can be too much of a good thing for some cells to deal with.

http://www.crseo.ucsb.edu/IOM2/Triple_Point.htm l.

Structure of Water Water is polar covalently bonded within the molecule. This unequal sharing of the electrons results in a slightly positive and a slightly negative side of the molecule. Other molecules, such as Ethane, are nonpolar, having neither a positive nor a negative side.

The difference between a polar (water) and nonpolar (ethane) molecule is due to the unequal sharing of electrons within the polar molecule. Nonpolar molecules have electrons equally shared within their covalent bonds.

Structure of Water Formation of a hydrogen bond between the hydrogen side of one water molecule and the oxygen side of another water molecule.

Water has been referred to as the universal solvent. Living things are composed of atoms and molecules within aqueous solutions (solutions that have materials dissolved in water).

Solutions are uniform mixtures of the molecules of two or more substances.

Structure of Water

The solvent is usually the substance present in the greatest amount (and is usually also a liquid). The substances of lesser amounts are the solutes.

Dissolution of an ionically bonded compound, sodium chloride, by water molecules.

Structure of Water Water tends to disassociate into H+ and OH- ions. In this disassociation, the oxygen retains the electrons and only one of the hydrogens, becoming a negatively charged ion known as hydroxide. Pure water has the same number (or concentration) of H+ as OHions. Acidic solutions have more H+ ions than OH- ions. Basic solutions have the opposite. An acid causes an increase in the numbers of H+ ions and a base causes an increase in the numbers of OH- ions.

pH scale The pH scale is a logarithmic scale representing the concentration of H+ ions in a solution.

If we have a solution with one in every ten molecules being H+, we refer to the concentration of H+ ions as 1/10.

Remember from algebra that we can write a fraction as a negative exponent, thus 1/10 becomes 10-1. Conversely 1/100 becomes 10-2 , 1/1000 becomes 10-3, etc.

Logarithms are exponents to which a number (usually 10) has been raised. For example log 10 (pronounced "the log of 10") = 1 (since 10 may be written as 101).

The log 1/10 (or 10-1) = -1. pH, a measure of the concentration of H+ ions, is the negative log of the H+ ion concentration.

If the pH of water is 7, then the concentration of H+ ions is 10-7.

pH scale

Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).

B- Organic molecules Organic molecules are those that: 1) formed by the actions of living things; and/or 2) have a carbon backbone. Methane (CH4) is an example of this. If we remove the H from one of the methane units below, and begin linking them up, while removing other H units, we begin to form an organic molecule.

Images from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).

B- Organic molecules Functional groups are clusters of atoms with characteristic structure and functions.

Polar molecules (with +/- charges) are attracted to water molecules and are hydrophilic.

Nonpolar molecules are repelled by water and do not dissolve in water; are hydrophobic.

Hydrocarbon is hydrophobic except when it has an attached ionized functional group such as carboxyl (acid) (COOH), then molecule is hydrophilic.

B- Organic molecules Isomers are molecules with identical molecular formulas but differ in arrangement of their atoms (e.g., glyceraldehyde and dihydroxyacetone).

Functional groups in organic molecules. Images from Purves et al., Life: The Science of Biology, 4th Edition,

B- Organic molecules Each organic molecule group has small molecules (monomers) that are linked to form a larger organic molecule (macromolecule). Monomers can be jouined together to form polymers that are the large macromolecules made of three to millions of monomer subunits.

Macromolecules are constructed by covalently bonding monomers by condensation reactions where water is removed from functional groups on the monomers.

C- Macromolecules There are four classes of macromolecules (polysaccharides, triglycerides, polypeptides, nucleic acids). These classes perform a variety of functions in cells.

Nucleic Acids: Deoxy-ribonucleic acid (DNA) Ribonucleic acid (RNA) Protein: Please, give an assay on protein “structure and classification”….thanks … due date next lecture.

Nucleic Acids Nucleic acids are polymers composed of monomer units known as nucleotides. There are a very few different types of nucleotides. The main functions of nucleotides are information storage (DNA), protein synthesis (RNA), and energy transfers (ATP and NAD). Nucleotides consist of a sugar, a nitrogenous base, and a phosphate. The sugars are either ribose or deoxyribose. They differ by the lack of one oxygen in deoxyribose. Both are pentoses usually in a ring form. There are five nitrogenous bases. Purines (Adenine and Guanine) are double-ring structures, while pyrimidines (Cytosine, Thymine and Uracil) are single-ringed.

Nucleic Acids

Structure of two types of nucleotide. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).

Nucleic Acids

Structure of a segment of a DNA double helix. Image from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates (www.sinauer.com) and WH Freeman (www.whfreeman.com).

Nucleic Acids

Changes in DNA information will be translated into changes in the primary structure of a polypeptide, and from there to the secondary and tertiary structures.

A mutation is any change in the DNA base sequence. Most mutations are harmful, few are neutral, and a very few are beneficial and contribute the organism's reproductive success. Mutations are the wellspring of variation, variation is central to Darwin and Wallace's theory of evolution by natural selection.

Early 1950’s: Rosalind Franklin, John Randall & Maurice Wilkins (King's College, London) made beautiful X-ray diffraction pictures of DNA fibers. Rosalind Franklin showed her DNA x-ray diffraction pictures to Watson and Crick. X-rays diffract X-ray source X-rays diffract

DNA DNA is often called the blueprint of life.

In simple terms, DNA contains the instructions for making proteins within the cell.

 Our

genes are on our chromosomes.

 Chromosomes

are made up of a chemical called DNA.

 DNA

is a very long polymer.

 The

basic shape is like a twisted ladder or zipper.

 This

is called a double helix.

Each cell has about 2 m of DNA. The average human has 75 trillion cells. The average human has enough DNA to go from the earth to the sun more The earth is 150 billion m than 400 times. or 93 million miles from the sun. DNA has a diameter of only 0.000000002 m.

What Kind of Chemical is DNA? To answer this question we must take a closer look at DNA. What is DNA made of? What does it look like?

DNA is a biopolymer •Polymers are molecules made of repeating units or building blocks •DNA has four chemical building blocks symbolized by the letters A,G,C,& T •The letters of your DNA are in a specific order that carries information about you!! So, a DNA polymer can be represented as a string of letters: A G CTT A G G GT A A A C C C AT AT A

Question: • What is DNA?

• The DNA double helix has two strands twisted together.

“Rungs of ladder” Nitrogenous Base (A,T,G or C)

“Legs of ladder”

Phosphate & Sugar Backbone

Phosphate Group

O O=P-O O

5

CH2

O N C1

C4 Sugar (deoxyribose)

C3

C2

Nitrogenous base (A, G, C, or T)



    

PURINES 1. Adenine (A) 2. Guanine (G)

PYRIMIDINES 3. Thymine (T) 4. Cytosine (C)

A or G

T or C



Base Purines Pyrimidines Pairs Adenine (A) Thymine (T) A = T



Guanine (G)



# of H-Bonds 2

Cytosine (C) C G

3

H-bonds

G

C

T

A



Adenine must pair with Thymine



Guanine must pair with Cytosine



Their amounts in a given DNA molecule will be about the same.

 If

there is 30% Adenine, how much Cytosine is present?

 There

would be 20% Cytosine.

 Adenine

(30%) = Thymine (30%)  Guanine (20%) = Cytosine (20%)  (50%) = (50%)

phosphate

• The backbone of the molecule is alternating phosphates and deoxyribose sugar • The teeth are nitrogenous bases.

deoxyribose

bases

DNA is a chemical contained in every cell of your body.

DNA is a Chemical!?

Yes!

What other kinds of chemicals are in your body? •We are made up of chemicals, formed from the elements carbon (C), hydrogen (H), oxygen (O), phosphorus (P) and others •DNA is made up of carbon (C), hydrogen (H), oxygen (O), phosphorus (P) •We breathe air which contains oxygen molecules (O2). •We eat food which is composed of chemicals called proteins, sugars, and fats. •Our bones are made up largely of calcium (Ca) •Our bodies make energy by breaking down chemicals such as sugars! •We store energy in our body in the form of carbohydrates.

DNA is a Biopolymer!? DNA POLYMER fun facts to know and trade: •Polymers are long molecules made up of repeated building blocks. •DNA polymers are always linear and are NOT branched. •DNA polymers can be very long and made up of millions of building blocks. •DNA building blocks are called bases. •DNA has 4 different types of building block bases called A,G,C,T

•DNA bases are chemicals that carry genetic information in their order.

5 min break

DNA Replication Question • When and where does DNA Replication take place?

 

“S” phase in interphase of the cell cycle. Nucleus of eukaryotes S phase

DNA replication takes place in the S phase.

G1

interphase

Mitosis -prophase -metaphase -anaphase -telophase

G2

• Origins of replication • 1. Replication Forks: hundreds of Yshaped regions of replicating DNA molecules where new strands are growing. 5’ Parental DNA Molecule 3’

3’

Replication Fork 5’



Origins of replication 2. Replication Bubbles: a. Hundreds of replicating bubbles (Eukaryotes). b. Single replication fork (bacteria). Bubbles

Bubbles

Strand Separation: 1. Helicase: enzyme which catalyze the unwinding and separation (breaking HBonds) of the parental double helix. 2. Single-Strand Binding Proteins: proteins which attach and help keep the separated strands apart.





Strand Separation: 3. Topoisomerase: enzyme which relieves stress on the DNA molecule by allowing free rotation around a single strand. Enzyme Enzyme

DNA

 



Priming: 1. RNA primers: before new DNA strands can form, there must be small preexisting primers (RNA) present to start the addition of new nucleotides (DNA Polymerase). 2.

Primase: enzyme that polymerizes (synthesizes) the RNA Primer.

• Synthesis of the new DNA Strands:

5’

1. DNA Polymerase: with a RNA primer in place, DNA Polymerase (enzyme) catalyze the synthesis of a new DNA strand in the 5’ to 3’ direction. 3’

Nucleotide

DNA Polymerase

RNA Primer

5’



Synthesis of the new DNA Strands:



2. Leading Strand: synthesized as a single polymer in the 5’ to 3’ direction.

5’

3’ 5’ Nucleotides

DNA Polymerase

RNA Primer

DNA Replication • Synthesis of the new DNA Strands: 3. Lagging Strand: also synthesized in the 5’ to 3’ direction, but discontinuously against overall direction of replication. Leading Strand

5 ’ 3’

DNA Polymerase

RNA Primer

3’ 5’

5’

3’

3’

5’ Lagging Strand

DNA Replication • Synthesis of the new DNA Strands: 4. Okazaki Fragments: series of short segments on the lagging strand. DNA Polymerase

Okazaki Fragment RNA Primer

5’ 3’

3’ 5’

Lagging Strand

• Synthesis of the new DNA Strands: 5. DNA ligase: a linking enzyme that catalyzes the formation of a covalent bond from the 3’ to 5’ end of joining stands. Example: joining two Okazaki fragments together. DNA ligase

5’ 3’

Okazaki Fragment 1

Lagging Strand

Okazaki Fragment 2

3’ 5’

• Synthesis of the new DNA Strands: 6. Proofreading: initial base-pairing errors are usually corrected by DNA polymerase.

• Semiconservative Model: 1. Watson and Crick showed: the two strands of the parental molecule separate, and each functions as a template for synthesis of a new complementary strand. DNA Template Parental DNA

New DNA

DNA Repair • Excision repair: 1. Damaged segment is excised by a repair enzyme (there are over 50 repair enzymes).

2. DNA polymerase and DNA ligase replace and bond the new nucleotides together.

•

DNA carries information-via the chemical “letters” of which it is made.

•

Therefore genes, which are made of DNA, contain information.

•

The information in genes constitutes “instructions” for making living things.

•

So where do we keep these instructions?

DNA double helix.--------------------DNA + Protein (histones) = Nucleosome.---------------------------Packed nucleosomes = Chromatin.-------------------------------

Folded section of chromosome--

Condensed folds----------------------

Duplicated chromosome-----------

Chromosome after DNA Replication.

Sister Chromatids = Identical daughter DNA molecules

 Virtually every cell in your body contains its own complete copy of all your DNA

 A single, complete copy of an organism’s DNA is called its genome

 The genome is a set of instructions, like a master plan, written in a molecular language, using DNA instead of paper and ink

 Therefore, each cell in your body has a copy of your genome, which is, in essence, a master plan for making you.

Your Genome is YOUR Body’s Master Plan! •ALL of the DNA instructions needed to make YOU! • All the parts needed to make a unique human. DNA

• Genes carry specific instructions to make parts. • Genes are written in DNA letters, A,G,C,T

Try to keep track of our genome components: • ~5% of genome encodes proteins • ~50% of genome contains transposable (movable) DNA elements that jump around the genome • ~50% of genome contains repeated sequences and DNA junk

MFH modified Human Genome Program, U.S. Department of Energy, Genomics and Its Impact on Medicine and Society: A 2001 Primer, 2001

 Electrophoresis:

a technique used to separate large molecules, based on the movement of charged particles in an electric field.

 Module

1:  DNA has a negative charge and as a result will move toward the positive electrode.  Module

2:  Use a molecular sieve to separate by size.

Gel is Made of Agarose: • Agarose is a chain-like molecule • Heat agarose in water to dissolve • Pour melted agarose into mold • Cool to make a Jello-like gel slab • Forms 3D molecular network

Gel Electrophoresis:  DNA loaded into wells  Gel subjected to electric field  DNA moves toward positive  (+) electrode  DNA must move through network made by agarose

Load DNA Samples into Wells

TOP

(-)

DNA Moves in Gel

TOP BOTTOM (-)

DNA Moves in Gel

(+)

BOTTOM

(+)

Gel Electrophoresis of reaction products

Thank You