Chapter 1 acid nucleic 2015813

ATP - MAJOR ENERGY SOURCE FOR CELLULAR ACTIVITY ATP: adenosine triphosphate  All cells need chemical energy carried out by ATP  Molecules in food store chemical energy in their bonds

CHAPTER 3

NUCLEIC ACID

GMO (Genetically modified organism)

GMF (Genetically modified food)

Human genome

NUCLEIC ACID - HISTORY

 Isolated nuclein (from nuclei of white blood cells)

Guanine(G) Thymine (T); Uracil (U) Cytocine (C)

NUCLEOTIDE STRUCTURE - SUBUNITS

NUCLEOTIDE STRUCTURE - BONDS

NUCLEOTIDE STRUCTURE - PENTOSE

THE SUGAR-PHOSPHATE BACKBONE

 Sugar-phosphate joined by phosphodiester bonds

 Sugar-phosphate in poly nucleotide orientated in the same

direction

 3’-OH group of the sugar in one nucleotide forms an ester bond to the phosphate group on the 5’-carbon of the sugar of the next

nucleotide

POLYNUCLEOTIDE-BACKBONE

NUCLEOTIDE STRUCTURE-BASE

Bases

 Attached to the 1 st Carbon

of sugar

NUCLEOSIDE & NUCLEOTIDE

• Nucleoside

– consists of a nitrogen base linked to C1’ of a ribose or

deoxyribose (glycosidic bond) – Named

• for purines: changing the nitrogen base ending: -ine to -

osine : adenine  adenosine

• for pyrimidines: changing the nitrogen base ending (osine to – idine: cytosine  cytidine

NUCLEOSIDE & NUCLEOTIDE

• Nucleotide

– nucleoside forms a phosphate ester with the C5’-OH group of ribose or deoxyribose

– Named: using the name of the nucleoside followed by

5’-monophosphate: adenosine 5’-monophosphate

replication)

NAMES OF NUCLEOSIDES AND NUCLEOTIDES

ATP - MAJOR ENERGY SOURCE FOR CELLULAR ACTIVITY

 ATP: adenosine triphosphate

 All cells need chemical energy carried out by ATP

 Molecules in food store chemical energy in their bonds

Starch molecule

Glucose molecule

HOW DOES ATP STORE ENERGY?

 Energy is stored in the last high energy phosphate

bond (it can store more energy than other types of

bonds)

 The cell stores energy by bonding a phosphate to

ADP (Adenosine Diphosphate)

ATP-CHEMICAL STRUCTURE

HOW DOES CELL GET ENERGY FROM ATP?: HYDROLYSIS

By breaking the high- energy bonds

between the last two

phosphates in ATP

H 2 O

THE ADP-ATP CYCLE

ATP & CHEMICAL ENERGY

Organisms break down carbon-based molecules to produce ATP

Carbohydrates: the most commonly broken down to make ATP

– not stored in large amounts– up to 36-38 ATP from one glucose molecule

 Fats store the most energy: 80 percent of energy in

the body– about 146 ATP produced from a triglyceride

 Proteins: the least likely to be broken down to make

ATP– amino acids not usually needed for energy

WHEN IS ATP MADE?

During Cellular Respiration

• Includes pathways that require oxygen

• Glucose is oxidized and O2 is reduced

• Glucose breakdown  one molecule of glucose 

36-38 ATP molecules

TYPES OF NUCLEIC ACID

DeoxyriboNucleic Acid (DNA)

contains genetic information of an organism

in the cell nucleus and mitochondria

RiboNucleic Acid (RNA)

 throughout the cell, much more abundant than DNA

 assisting in the expression of DNA to protein

DNA STRUCTURE – DISCOVERY

Rosalind Elsie Franklin(1920-1958),

British biophysicist and X-ray

Zealand-DNA STRUCTURE – DISCOVERY

James Dewey Watson (1928)

American molecular biologist, geneticist, zoologist

Francis Harry Compton Crick

(1916-2004), English molecular biologist, biophysicist, and neuroscientist

Watson and Crick with their DNA model

 Francis Crick and James Watson working at the Cavendish

Laboratory in Cambridge (1953), discovered the structure of

DNA, a double helix

DNA STRUCTURE – THE NOBEL PRIZE

 Crick, Watson and Wilkins won the Nobel

Prize for medicine in 1962

 Rosalind Franklin, Maurice Wilkins’s

colleague, developed the technique to

photograph a single strand of DNA  died

of cancer in 1958 could not be recognized

in the Nobel Award

WATSON & CRICK MODEL OF DNA

Two strands of polynucleotides

 wind together

 run in opposite directions

(antiparallel)

 Complementary

WATSON & CRICK MODEL OF DNA

Nucleotides bond between DNA

 Arranged in step-like pairs

 Determines the genetic

information of DNA

WATSON & CRICK MODEL OF DNA

Ratio of A-T : G-C affects stability

of DNA molecule

 Biotech procedures

more G-C = need higher T°

to separate strands

BASE-PAIRING RULE OF NUCLEOTIDE

Erwin Chargaff (1905-2002),

Austrian American biochemist, discovered the base-pairing rule of nucleic acids

Purine – Pyrimidine pairing

A :: T (2 H bonds)

G ::: C (3 H bonds)

ERWIN CHARGAFF’S DNA DATA (1950-51)

DNA - FUNCTION

Storage of genetic information

Self-duplication & inheritance

Expression of the genetic message

Different arrangement of nucleotides in DNA  biodiversity

DNA - SEQUENCE

Reading DNA sequence

 The sequence: read from 5’ to 3’

end using the letters of the bases

• Ex: 5’—A—C—G—T—3’

 Free 5’ end: phosphate group

 free 3’ end: - OH group

DNA - THE DOUBLE HELIX

Base pairs of two strands: consists of

a purine and a pyrimidine  the same

width, keeping the two strands at

equal distances from each other

one helical turn

34 Å

major groove

12 Å

minor groove

6 Å

DNA - SUPERCOILS

Each cell contains about 2 meters

of DNA  DNA “packaged” by

coiling around a core of proteins

(histones: rich in lysine

and arginine residues)

The DNA-histone: nucleosome

In eukaryotic cells (animals,

plants, fungi), DNA stored in the

nucleus

Properties of DNA

D DNA can be renatured

1 Effect of time, conentration and complexity on renaturation

E General forms of a DNA helix

1 A form

2 B form

3 Z form

DNA DENATURATION

DENATURATION & RENATURATION OF DNA

FORMS OF A DNA HELIX

Structure and Function correspondence

of protein and nucleic acid

Fibrous protein Globular protein Helical DNA Globular RNA

Structural protein  Enzymes

 Antibodies

 Receptors, etc

Genetic information maintenance

 Ribosomal RNA (rRNA)

 Transfer RNA (tRNA)

 Signal recognition

Chemical and physical properties of DNA

• Stability

• Effect of acid and alkali

• Chemical denaturation

• Viscosity

1 Hydrogen bonding

• Stability lies in the stacking interactions between base

pairs

2 Stacking interaction/hydrophobic interaction between

aromatic base  maximized in double-stranded DNA

(base stacking & hydrophobic effect)

Effect of Acid

• Strong acid (pH3-4) + high temperature (perchloric acid+100C)  completely hydrolyzed to bases, riboses/deoxyribose, and

phosphate

 Maxam and Gilbert chemical DNA sequencing

DNA sequencing technique based on chemical removal and

modification of bases specifically and then cleaving the

sugar-phosphate backbone of the DNA and RNA at particular bases

Effect of Alkali

• DNA denaturation at high pH

Base pairing is not stable anymore because of the change of

tautomeric (states of the bases DNA denaturation

RNA is hydrolyzed at higher pH because of 3’-OH groups in RNA

Chemical DenaturationFormamide (HCONH2) and Formaldehyde : Northern blot

Disrupting the hydrogen bonding of the bulk water solution

Hydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structure

Reasons for the DNA high viscosity

1 High axial ratio

2 Relatively stiff

Applications

1 Long DNA molecules can easily be shortened by

shearing force

2 When isolating very large DNA molecule, always

avoid shearing problem

Rapid cooling

Slow cooling Whole complementation of dsDNA

Annealing Base paring of short regions of complementarity

within or between DNA strands

(example: annealing step in PCR reaction)

Hybridization Renaturation of complementary sequences between different nucleic acid molecules

(examples: Northern or Southern hybridization)

Buoyant density

Purifications of DNA: equilibrium density gradient centrifugation

RNA pellets at the bottom

Protein floats

Spectroscopy of Nucleic Acid

1 UV absorption

- Nucleic acids absorb UV light due to the aromatic bases

- The wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax = 260 nm)

- Applications: detection, quantitation, assessment of purity (A260/A280)

Quantitation of nucleic acid

Extinction coefficient (e): 1 mg/ml dsDNA has an A260 of

20 ssDNA and 25 RNA

The values for ssDNA and RNA are approximate

- (e : purines > pyrimidines)

DIFFERENCES BETWEEN RNA & DNA

RNA (ribonucleic acid)

 Differences between RNA and DNA

- Pentose sugar: ribose, [deoxyribose in DNA]

- Uracil replaces thymine

- Single stranded [DNA: double stranded]

- Much smaller than DNA

- Three main types of RNA:

ribosomal (rRNA), messenger (mRNA) and transfer (tRNA)

RNA STRUCTURE

TYPES OF RNA

RIBOSOMAL RNA (rRNA) & MESSENGER RNA (mRNA)

Ribosomal RNA

 65% of Ribosomes ( Sites of protein synthesis)

Messenger RNA

 Carries the genetic code to ribosomes

 Complementary to the DNA of the gene)

TRANSFER RNA (tRNA)

Transfer RNA

 Translates the genetic code from the mRNA

 Brings specific amino acids to the ribosome for protein synthesis

 Each amino acid is recognized by one or more specific tRNAs

- one end: attaches to the amino acid

- the other end binds to the mRNA (complimentary sequence)

GENETIC CODE

GENETIC CODE ORGANIZATION

 Single-base changes (single-nucleotide polymorphism)

in the third position in a codon  produce the same amino acid

 The second base specifies if the amino acid is polar or

apolar (hydrophobic)

 Changes elsewhere in the codon  produce a different

amino acid, but with the same physical-chemical

properties

Polar R groups make the amino acid hydrophilic

Non-polar R groups make the amino acid hydrophobic

Ionic R groups make the amino acid hydrophilic

READING THE GENETIC CODE

Ex: determine the amino acid sequence coded

by a section of a mRNA

5’—CCU —AGC—GGA—CUU—3’

According to the genetic code

 amino acids sequence

CCU = Proline AGC = Serine GGA = Glycine CUU = Leucine

mRNA section codes for the amino acid

sequence

Pro—Ser—Gly—Leu

THE CENTRAL DOGMA OF MOLECULAR BIOLOGY (BY F CRICK)

 Replication: DNA is copied with very high fidelity

 Transcription: DNA genetic code is read and transferred to

messenger RNA (mRNA)

 Translation: genetic code is converted to a protein

Francis Harry Compton Crick (1916-2004)

WHEN DO CELLS REPLICATE THEIR DNA?

Preparation for mitosis (for growth: daughter

cells have an identical copy of the DNA)

Preparation for meiosis (reduce the number of chromosomes present in each gamete  Sex cells copy their DNA)

Repair, replace dead cells

Have enough DNA available to act as a template for RNA during transcription

DNA REPLICATION - OVERVIEW

DNA Replication - Semi-Conservative

Reiji Okazaki (1930-1975)

Japanese molecular biologist

 Discovered Okazaki fragments with his wife in 1966

Okazaki fragments

• 1,000-2,000 nu long in E coli

• 100-200 nu long in eukaryote

• separated by ~10 nu RNA primers

ACCURACY OF DNA REPLICATION

A mismatching of base pairs can

occur at a rate of 1 per 10,000 bases

Chances of a mutation: occurring at any gene: about 1 in 10,000

DNA polymerase proofreads and

repairs accidental mismatched pairs

PROOFREADING AND REPAIRING DNA

Nuclease

DNA

polymerase

DNA ligase

A thymine dimer distorts the DNA molecule.

2

Repair synthesis by

a DNA polymerase fills in the missing nucleotides.

unwinding and other processes

 lead to the cessation of DNA replication and transcription

 cause the cross-linking of DNA double strand and DNA with protein

DNA replication blocked  cell death

Application: ultraviolet sterilization  damage DNA of bacteria

(disturbing DNA replication)

 rupture of phosphodiester bond  DNA breaks

Application: radiation therapy of cancer: using ionizing radiation

 damage the DNA of cancer cells stop its division  cell death

DNA DAMAGE

Chemical factors

base molecular isomerization  change position of hydrogen bonds between bases

bases mismatched in the replication

O2, H2O2, chemicals yielded by biological metabolism

heavy metal ions, medicine, pesticide, and their

metabolites

DNA DAMAGE

Chemical factors

deamination damage of DNA bases: cytosine, adenine and guanine bases with exocyclic-amino (-NH2)  removed under the influence

of water, oxidants and free radicals and some other substances

turning the cytosine into uracil,…

Error in the replication (bases-mismatched)  gene mutation

 Nitrite induce the deamination of adenine to form

hypoxanthine

 paired with cytosine in the DNA replication  resulting in A • T

→ G • C conversion

Depending on nucleotide change

not change aa sequence (many codon encode for 1 aa)

AUG GGU AGG GAG  AUG GGU AG A GAG

Arg Arg

Change start codon  protein not made AUG GGU AGG GAG  AU U GGU AG A GAG

 Frame shift mutation

Insertions or deletions of nucleotides

Change reading frame

Insertions

AUG GGU AGG GAG GCA ACC UGA ACC GAC

AUG GGU AGG A GA GGC AAC CUG AAC CGA C

 DeletionsAUG GGU AGG GAG GCA ACC UGA ACC GAC

 AUG GGU GGG AGG CAA CCU GAA CCG AC

Other mutations

 Stop codon inserted  truncated protein

AUG GGU AGG GAG GCA ACC UGA ACC GAC

 AUG GGU AGG GAG GCA ACC UGA TAA ACC GAC

 Stop codon changed  extra long protein

 AUG GGU AGG GAG GCA ACC UGA ACC GAC TAA

 AUG GGU AGG GAG GCA ACC UGA ACC GAC TAT

GENETIC ENGINEERING

 Addition, deletion, or manipulation

of a single trait in an organism to

create a desired change

 Major tool: recombinant DNA

GENETIC ENGINEERING (GE)

Manipulating DNA

• DNA Extraction – chemical procedure separate DNA

• DNA Cutting – restriction enzymes cut particular DNA sequences

• Separating DNA – gel electrophoresis

• Copy – using polymerase chain reaction “PCR”

• Recombinant DNA

• Genetically Modified Organism (GMO) (Transgenic

Organism)

• Plant DNA + bacterial DNA

 prevent disease for plant

• Human gene + goat DNA  for blood clotting agent production

• Insert a foreign gene into a host: Plasmid into bacterial cell – transformation or transfection-organism (transgenic ( eukaryote) or

GENETIC ENGINEERING - BENEFITS

 Creates new crops and farm animals (crops

grows in desert heat, or without fertilizer)

 Make bacteria that can make medicines,

chemicals

 Grow human body parts

 Prevent genetic diseases

 Food shelf-life: extended; storage,

handling: simplified

 Resist spoilage

 Plants: disease resistant

 Animals: produce hormones, more milk, leaner meat

 Alternating growth hormones

HARM

TOOLS FOR GENETIC ENGINEERING

GEL ELECTOPHORESIS

POLYMERASE CHAIN REACTION (PCR)

Amplifying DNA using:

 DNA polymerase,

Taq: low replication fidelity (error rate: 1 in 9000 nu.),

no proof reading activity

Pfu: proof reading activity

 over one million copies per original

Kary Banks Mullis (1944)

American biochemist, Nobel Prize for his invention of PCR method

PCR

Introducing free DNA into bacteria

Transformation- process of introducing free DNA into bacteria

 Competent cell- a cell that is capable of taking

up DNA

 Heatshock

 Electroporation- use of an electric shock to

momentarily open or disrupt cell walls

Conjugation- the contact of bacteria that involves the exchange of DNA with a mating tube

Transduction: injection of foreign DNA by a

bacteriophage virus into the host bacterium

GENETICALLY MODIFIED ORGANISM (GMO)

• Transgenic organisms (gmo) contain genes

from other organisms

• A clone: member of a population of

genetically identical cells produced from a

single cell