Biochemistry

Lecture 6

Functions ofNucleotides and Nucleic

Acids• Nucleotide Functions:

– Energy for metabolism (ATP)– Enzyme cofactors (NAD+)– Signal transduction (cAMP)

• Nucleic Acid Functions: – Storage of genetic info (DNA)– Transmission of genetic info (mRNA)– Processing of genetic information (ribozymes)– Protein synthesis (tRNA and rRNA)

NucleotideNucleosideNucleobase

Pyrimidine Nucleobases

Purine Nucleobases

UV Absorption of Nucleobases

-D-ribofuranose in RNA

-2’-deoxy-D-ribofuranose in DNA

N-Glycosidic Bond

Polynucleotides

Hydrolysis of RNA

Hydrogen Bonding!

Discovery of DNA Structure

• One of the most important discoveries in biology

• Why is this important– "This structure has novel features which are of

considerable biological interest“--- Watson and Crick, Nature, 1953

• Good illustration of science in action:– Missteps in the path to a discovery– Value of knowledge– Value of collaboration– Cost of sharing your data too early

Covalent Structure of DNA (1868-1935)

• Friedrich Miescher isolates “nuclein” from cell nuclei

• Hydrolysis of nuclein:– phosphate– pentose– and a nucleobase

• Chemical analysis:– phosphodiester linkages– pentose is ribofuranoside

O

OH

H HH

Thymine

H

CH2O P

OH

O

O

P OOH

OH

O

H

H HH

Adenine

H

CH2O P

OH

O

O

Structure of DNA: 1929

(Levene and London)

Structure of DNA:

1935(Levene and Tipson)

C5H7OThymine

O

P

O

POH

OH

O

O

O

OH

C5H7OAdenine

O

Road to the Double Helix• Franklin and Wilkins:

–“Cross” means helix

–“Diamonds” mean

that the phosphate-

sugar backbone

is outside

– Calculated helical

parameters

• Watson and Crick:

– Missing layer means

alternating pattern

(major & minor groove)

– Hydrogen bonding:

A pairs with T

G pairs with C

Double helix fits the data!

Watson, Crick, and Wilkins shared 1962 Nobel Prize

Franklin died in 1958

Other forms of DNA

The Central Dogma

DNA Replication

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material”

Watson and Crick, in their Nature paper,1953

Using DNA Structure

Thermal Denaturation

Molecular Mechanisms of Spontaneous Mutagenesis

• Deamination• Very slow reactions• Large number of residues• The net effect is significant: 100 C U events /day in a mammalian cell

• Depurination• N-glycosidic bond is hydrolyzed• Significant for purines: 10,000 purines lost/day in a mammalian cell

• Cells have mechanisms to correct most of these modifications.

DNA Technologies

DNA Cloning

Restriction Enzymes

Antibiotic Selection• Antibiotics, such as penicillin and ampicillin, kill

bacteria

• Plasmids can carry genes that give host bacterium a resistance against antibiotics

• Allows growth (selection) of bacteria that have taken up the plasmid

NH

S

N

N

OO

H

OOH

HH

PCR

PolymeraseChainReaction

Site-Directed Mutagenesis

DNA Electrophoresis

DNA Sequencing

DNA Sequencing

Shotgun Sequencing

DNA Fingerprinting

Expression of Cloned Genes

Protein Purification

Eukaryotic Gene Expression in Bacteria

• An eukaryotic gene from the eukaryotic genome will not express correctly in the bacterium

• Eukaryotic genes have

– Exons: coding regions

– Introns: noncoding regions

• Introns in eukaryouric gene pose problems

• Bacteria cannot splice introns out

• mRNA is intron-free genetic material

cDNA

DNA Microarrays: Applications

DNA Microarrays allow simultaneous screening of many thousands of genes: high-throughput screening

• genome wide genotyping

– Which genes are present in this individual?

• tissue-specific gene expression

– Which genes are used to make proteins?

• mutational analysis

– Which genes have been mutated?

DNA Microarrays: Design Two fundamental approaches• One-color array

– Patented and commerialized by Affymetrix– Photolitographic synthesis of probe DNA on the chip– Targets are biotin labeled– Bound targets detected using streptavidin-fluorofore complex– Widely used in industry

• Two-color array– Developed by Stanford University, 1996– Probes sometimes pipetted on the chip– Targets linked to either green or red fluorescent labels– Used often in academia